The electrochemical activity of the carbon nanotube was utilized to modulate the drug release. The release of ibuprofen from a novel hybrid hydrogel composed of sodium alginate (SA), bacterial cellulose (BC), and multi-walled carbon nanotubes (MWCNTs) was studied by Shi et al. [176]. The release of ibuprofen can be provoked by electrostatic repulsion. Thereby, the on–off release mechanism can be attained by introducing electrochemical potential [176].

#### **9. Conclusions**

Modified and functionalized nanocelluloses with low toxicity and high biocompatibility render them promising materials as advanced drug carriers. Various hydroxyl groups on the surface of the nanocellulose serve as attachment sites of drugs through covalent and/or physical interactions. In addition, nanocellulose modification results in a different morphological structure for the carrier, which contributes to an increase in the diffusion pathway of the drug within the carrier. Therefore, surface chemistry is a crucial factor that should be considered in the design of nanocellulose as a drug carrier for effective drug delivery. High-purity nanocelluloses are also required to obtain drug carriers with the well-constructed framework, thus facilitating drug adsorption and release control. Considering all these factors, carrier-based nanocellulose is a promising candidate for developing novel sustained drug delivery systems.

**Author Contributions:** Conceptualization V.B.L., F.E.S. and S.I.; redraw the figures V.B.L. and M.Y.; drafting the manuscript V.B.L., J.N.P., S.P.S. and S.I.; Supervision S.I. and F.E.S.; review and editing, J.S, and Y.H.J.; proof reading J.S. and Y.H.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Directorate of Research and Community Service, Deputy for Strengthening Research and Development, Ministry of Research and Technology/National Research and Innovation Agency, Number: 150J/WM01.5/N/2021.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

*Article*

## **Isolation and Characterization of Nanocellulose with a Novel Shape from Walnut (***Juglans Regia* **L.) Shell Agricultural Waste**

**Dingyuan Zheng 1,2 , Yangyang Zhang 1,2, Yunfeng Guo 1,2 and Jinquan Yue 1,2,\***

Received: 3 June 2019; Accepted: 24 June 2019; Published: 3 July 2019

**Abstract:** Herein, walnut shell (WS) was utilized as the raw material for the production of purified cellulose. The production technique involves multiple treatments, including alkaline treatment and bleaching. Furthermore, two nanocellulose materials were derived from WS by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) oxidation and sulfuric acid hydrolysis, demonstrating the broad applicability and value of walnuts. The micromorphologies, crystalline structures, chemical functional groups, and thermal stabilities of the nanocellulose obtained via TEMPO oxidation and sulfuric acid hydrolysis (TNC and SNC, respectively) were comprehensively characterized. The TNC exhibited an irregular block structure, whereas the SNC was rectangular in shape, with a length of 55–82 nm and a width of 49–81 nm. These observations are expected to provide insight into the potential of utilizing WSs as the raw material for preparing nanocellulose, which could address the problems of the low-valued utilization of walnuts and pollution because of unused WSs.

**Keywords:** Walnut shell; nanocellulose; TEMPO oxidation; sulfuric acid hydrolysis; ultrasonication

## **1. Introduction**

With the depletion of fossil fuels and the increase in ecological and environmental problems caused by the usage of fossil fuels, the development and utilization of green biomass-based materials derived from renewable natural resources have been extensively studied around the world [1]. Cellulose, which can be observed in all the plant structures, is a common example of a renewable natural resource and offers the advantages of abundant availability, renewability, and biodegradability [2,3]. With the development of nanotechnology, nanocellulose, prepared from cellulose, has attracted significant attention from academic and industrial researchers because of its low cost, biocompatibility, biodegradability, nontoxicity, renewability, sustainability, strong surface reactivity, and desirable physical properties (it is lightweight and impermeable to gas and it also exhibits high stiffness, good optical transparency, and low thermal expansion) [4,5]. Compared with cellulose, nanocellulose exhibits large surface area, high crystallinity, high mechanical strength, high hydrophilicity and supramolecular structure [6]. These characteristics make nanocellulose promising for various applications such as polymer nanocomposites [7,8], packaging [9,10], electronics [11,12], and stimulus-responsive materials [13,14].

Nanocellulose can be extracted from several cellulose resources such as wood [15,16], cotton [17,18], ramie [19], bagasse [20,21], bamboo [22], sisal [23], corn straw [24], rice straw [18], and coconut shell [25]. It has been reported that the fundamental properties of the obtained nanocellulose, such as morphology, crystallinity, dimensions, and surface chemistry, vary highly depending on the raw material and the isolation process used to obtain it [26,27]. Hence, different categories of nanocellulose, such as cellulose nanofibrils, cellulose nanocrystals, and bacterial cellulose, which differ in terms of their dimensions and morphologies, exist [2,28]. These key properties are of critical importance for ensuring the end use of the isolated nanocellulose.

Walnut (*Juglans regia* L.), also known as a peach, is harvested from the seed of the walnut tree. Walnut is one of the world's four major "dried fruits" along with almonds, cashews, and hazelnuts. The global walnut production in 2017 exceeded 3.8 million tons. The walnut shell (WS) is the hard outer shell (endocarp) of walnut, which comprises cellulose, hemicellulose, lignin, and other small molecular substances and accounts for 67% of the total weight of walnut [29]. China, which is the top walnut producer in the world, produced 1.92 million tons of walnuts in 2017, which resulted in approximately 1.29 million tons of WSs (UN Food and Agriculture Organization, Corporate Statistical Databas, 2018). After the nut is removed from the WS, the shells are considered to be agricultural and forestry waste, which is often burned as fuel, seriously damaging the environment. Only a small portion of the produced WSs is used for preparing wood–plastic composite materials [30], carbon materials [31,32], or handcrafted products. Thus, the utilization of WSs is not much valued. The walnut contains desirable substances such as cellulose, hemicellulose, and lignin. It has been reported that the cellulose content of walnut shell is about 22% [33]. Hence, in this study, we investigate the preparation of nanocellulose using WS as the raw material for the first time.

Cellulose nanofibrils are mainly obtained from cellulosic fibers by mechanical treatments such as high-pressure homogenization, microfluidization, grinding, and ultrasonication [34]. However, such mechanical fibrillation methods are very energy intensive. Various pretreatment methods (such as enzymatic hydrolysis [35], 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation [36–38]) have been proposed to reduce the energy required for the mechanical deconstruction process by reducing the negative or positive charge on the fiber surfaces and by enhancing the colloidal stability of the final cellulose nanofibrils.

Unlike cellulose nanofibrils, cellulose nanocrystals have a rod-like morphology [39]. Cellulose nanocrystals are generally prepared by hydrolysis using a strong inorganic acid such as sulfuric acid [40,41], other inorganic strong acid hydrolysis are used for cellulose nanocrystals preparation as well, including hydrochloric acid, phosphoric acid, hydrobromic acid, and nitric acid [42]. Recent technological developments have resulted in a few sustainable and environment friendly methods that rely on recyclable chemicals. Examples include hydrolysis with solid acids (e.g., phosphor tungstic acid [43]) or treatment with ionic liquids [44] or deep eutectic solvents [45]. Among these, sulfuric acid is the most commonly used acid for producing sulfonated cellulose nanocrystals exhibiting good dispersibility in water [42]. During the hydrolysis process, the paracrystalline or disordered parts of cellulose are hydrolyzed and dissolved in the acid solution; however, the crystalline parts are chemically resistant to the acid and remain intact. Consequently, the cellulose fibrils are transversely cleaved, yielding short cellulose nanocrystals with a relatively high crystallinity [34].

In this study, we aim to isolate nanocellulose from a renewable, cheap, and currently underutilized raw material, i.e., WS. Herein, WS is pretreated using several mechanical and chemical processes, including grinding, extraction, alkali treatment, and bleaching. Nanocellulose was produced by TEMPO oxidation accompanied by ultrasonic treatment (yielding TNC) or sulfuric acid hydrolysis followed by ultrasonication (SNC). The resulting TNC and SNC and the intermediate products were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). The proposed method of extracting nanocellulose from WSs is expected to reduce the pressure on natural resources. This will offer an alternative high-valued utilization for the currently wasted WSs and, furthermore, to serve as a reference for future studies to improve the utilization of the obtained nanocellulose for developing new biobased nanomaterials.

#### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. Materials 2.1. Materials*

The WS collected from the Shanxi Province in China was used as the raw material. Ethanol, sodium hydroxide, sodium chlorite, sodium bromide, sodium hypochlorite, glacial acetic acid, sulfuric acid, and other chemicals were of analytical grade and used without any further purification. All of the chemicals were supplied by Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Distilled water was used the whole process. TEMPO was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Dialysis bags (MD44, viskase, Lombard, IL, USA) were provided by Beijing Biotopped Science & Technology CO., Ltd. (Beijing, China). The WS collected from the Shanxi Province in China was used as the raw material. Ethanol, sodium hydroxide, sodium chlorite, sodium bromide, sodium hypochlorite, glacial acetic acid, sulfuric acid, and other chemicals were of analytical grade and used without any further purification. All of the chemicals were supplied by Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Distilled water was used the whole process. TEMPO was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Dialysis bags (MD44, viskase, Lombard, IL, USA) were provided by Beijing Biotopped Science & Technology CO., Ltd. (Beijing, China).

#### *2.2. Preparation of Nanocellulose 2.2. Preparation of Nanocellulose*

The procedure used to prepare nanocellulose is outlined in Figure 1. The procedure used to prepare nanocellulose is outlined in Figure 1.

**Figure 1.** Procedure used to isolate nanocellulose from the walnut shell. **Figure 1.** Procedure used to isolate nanocellulose from the walnut shell.

#### 2.2.1. WS Pretreatment 2.2.1. WS Pretreatment

In natural WSs, cellulose is embedded in a network structure in which lignin and hemicellulose are linked together, mainly by covalent bonds [46]. Thus, WS was initially pretreated to isolate the nanocellulose. Pretreatment of walnut shell was performed using the method described by de Rodriguez, Thielemans, and Dufresne [47]. The WS was ground and sieved through 60 mesh (0.25 mm), dried in an oven at 105 °C, and stored in a desiccator. To eliminate hemicellulose, the obtained WS powder was mixed with a 2 wt.% NaOH solution in a WS/NaOH solid-to-liquid ratio of 10 g/100 mL and stirred for 4 h at 100 °C. This NaOH treatment was repeated four times until no more discoloration occurred; the product obtained after the NaOH treatment can be referred to as N-WS. Further, N-WS was bleached in a solution containing equal amounts of acetate buffer and 1.7 wt.% NaClO2 at a WS/solution solid-to-liquid ratio of 5 g/100 mL for 6 h at 80 °C (the reaction flask was shaken every 20 min to ensure the reaction occurs evenly). This bleaching process was repeated four times. The bleached products were then thoroughly washed using distilled water. The obtained product can be referred to as B-WS. In natural WSs, cellulose is embedded in a network structure in which lignin and hemicellulose are linked together, mainly by covalent bonds [46]. Thus, WS was initially pretreated to isolate the nanocellulose. Pretreatment of walnut shell was performed using the method described by de Rodriguez, Thielemans, and Dufresne [47]. The WS was ground and sieved through 60 mesh (0.25 mm), dried in an oven at 105 ◦C, and stored in a desiccator. To eliminate hemicellulose, the obtained WS powder was mixed with a 2 wt.% NaOH solution in a WS/NaOH solid-to-liquid ratio of 10 g/100 mL and stirred for 4 h at 100 ◦C. This NaOH treatment was repeated four times until no more discoloration occurred; the product obtained after the NaOH treatment can be referred to as N-WS. Further, N-WS was bleached in a solution containing equal amounts of acetate buffer and 1.7 wt.% NaClO2 at a WS/solution solid-to-liquid ratio of 5 g/100 mL for 6 h at 80 ◦C (the reaction flask was shaken every 20 min to ensure the reaction occurs evenly). This bleaching process was repeated four times. The bleached products were then thoroughly washed using distilled water. The obtained product can be referred to as B-WS.

### 2.2.2. TNC Preparation

TNC was prepared using the method described by Kuramae, Saito, and Isogai [48], with few modifications. One gram of B-WS was dispersed in 100 mL of distilled water; further, 0.1 g of sodium bromide and 0.016 g of TEMPO were added, followed by 20 mmol of NaClO. During the TEMPO oxidation, the pH of the suspension was adjusted to 10 ± 0.5 using 0.1 M NaOH and 0.1 M HCl. The reaction was continued for 5 h at room temperature under magnetic stirring at 1000 rpm and was terminated by adding 10 mL of ethanol. The oxidized B-WS was washed and filtered; subsequently, it was stored with distilled water at 4 ◦C to avoid strong hydrogen bonding.

To convert the nonoxidized hydroxyl and aldehyde groups in the oxidized B-WS into carboxyl groups, the product obtained in the previous step was further processed. One gram of oxidized B-WS (dry weight) was dispersed in 65 mL of distilled water, and the pH value was adjusted to 4–5. Further, 0.6 g of NaClO2 was added to the reaction system. The reaction was allowed to proceed for 1 h at 70 ◦C under magnetic stirring at 1000 rpm to yield carboxylated B-WS. The obtained carboxylated B-WS had a carboxylate content of 1.12 mmol/g as determined by conductivity titration [49]. A certain amount of carboxylated B-WS was weighed, and the mass fraction was adjusted to 0.5 wt.%. Finally, high-intensity ultrasonication was performed in an ultrasonic cell pulverizer (SCIENTZ-1200E, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) at 600 w for 30 min in an ice/water bath to yield TNC.

#### 2.2.3. SNC Preparation

SNC was prepared using the method described by Beck-Candanedo [41]. One gram of B-WS was added to 8.75 mL of the sulfuric acid solution (64 wt.%) under vigorous stirring, and the hydrolysis reaction was allowed to proceed for 1 h at 45 ◦C. The reaction was ended by adding distilled water in a volume that was ten times the reaction volume. The obtained suspension was washed by mixing with distilled water followed by centrifugation at 12,000 rpm for 15 min to eliminate the excess acid; the washing was repeated until the precipitate generation was terminated. The suspension was subsequently dialyzed against distilled water until the pH became 6.5–7. The dialyzed suspension was sonicated at 600 w for 2 min to yield SNC.

#### *2.3. Characterization of TNC, SNC, and Intermediate Products*

#### 2.3.1. Analysis of the Chemical Components

The α-cellulose content was determined as follows [50]. Two grams of WS powder was weighed and transferred to a 250-mL Erlenmeyer flask, and 25 mL of nitric acid/ethanol solution (1:4 by volume) was added. The mixed solution was refluxed for 1 h, and the process was repeated several times until the sample turned white. The powder was repeatedly washed using distilled water and filtered until the pH became neutral; the obtained residue was dried at 105 ◦C. The α-cellulose content (*X*, %) was calculated as follows

$$X = \frac{G}{G\_1(1-W)} \times 100\tag{1}$$

where *G* denotes the weight of the obtained residue, *G1* denotes the weight of the WS, and *W* denotes the moisture content of the WS.

The remaining chemical components of WS (organic extracts, lignin, hemicellulose, ash, and holocellulose) were analyzed according to the Technical Association of Pulp and Paper Industry standards that have been previously described [51].

#### 2.3.2. Scanning Electron Microscopy

The WS, N-WS, B-WS, TNC, and SNC microstructures were observed using a scanning electron microscope (SU8010, Hitachi, Japan) at an accelerating voltage of 5.0 kV. TNC and SNC were freeze-dried before observation. All the samples were coated with gold.

### 2.3.3. Transmission Electron Microscopy

TNC and SNC were imaged using a transmission electron microscope (H-7650 Hitachi, Japan) at a 100-kV acceleration voltage. The TNC and SNC suspensions were diluted to a concentration of 0.01% and deposited onto carbon-coated grids (230 mesh, Beijing Zhongjingkeyi Technology Co., Ltd., Beijing, China). After drying, the samples were negatively stained using a 1% phosphotungstic acid solution for 10 min. The TEM images were analyzed using Nanomeasurer 1.2 (Department of Chemistry, Fudan Univ., Shanghai, China) to determine the TNC and SNC size distributions.

#### 2.3.4. Fourier-transform Infrared Spectroscopy

The FTIR spectra of WS, N-WS, B-WS, TNC, and SNC were recorded on a Fourier-transform infrared instrument (Nicolette 6700, Thermo Fisher Scientific Inc., Waltham, MA, USA) in 400–4000 cm−1 with a resolution of 4 cm−1 , and 20 scans for each sample were conducted. The FTIR spectra of all samples were collected using the attenuated total reflection technique (ATR, the ATR crystal material is zinc selenide (ZnSe)).

### 2.3.5. X-ray Diffraction Technique

XRD analysis was performed on WS, N-WS, B-WS, TNC, and SNC using a diffractometer (D/max 2200, Rigaku, Japan) equipped with Ni-filtered Cu Kα radiation (λ = 1.5406 Å) at 40 kV and 30 mA. The diffraction intensities were recorded in 2θ = 5 ◦–60◦ with a scan rate of 5◦ /min.

The crystallinity index (CrI, %) was calculated according to the method reported by Segal [52] as follows

$$\text{CrI} = \frac{I\_{200} - I\_{am}}{I\_{200}} \times 100 \tag{2}$$

where *I200* is the maximum intensity of the diffraction at 200 peak (2θ = 22.6◦ ) and *Iam* is the intensity of the diffraction at 2θ = 18◦ .

#### 2.3.6. Thermogravimetric Analysis

The thermal stability of each sample was evaluated using a thermogravimetric analyzer (TG209F1, Netzsch Scientific Instruments Trading (Shanghai) Co., Ltd., Shanghai, China) from room temperature to 600 ◦C at a rate of 10 ◦C /min in a nitrogen atmosphere.

#### **3. Results and Discussion**

#### *3.1. Chemical Components*

Table 1 presents the WS, N-WS, and B-WS chemical compositions. The chemical compositions of the three samples were observed to be significantly different because of the applied chemical treatments.

**Table 1.** Chemical components of walnut shell WS, walnut shell treated by NaOH (N-WS), and walnut shell after bleaching (B-WS).

It is found that WS has the lowest percentage of cellulose and highest percentage of noncellulosic components such as lignin and hemicelluloses. The chemical treatments aim to remove the noncellulosic components. When WS was subjected to NaOH treatment, the lignin and hemicelluloses contents of N-WS decreased to 30.98% and 7.6%, respectively, whereas the cellulose concentration of N-WS increased to 56.6%. After bleaching treatment, the cellulose content of B-WS increased to 87.9% due to the removal of remaining lignin and hemicelluloses, resulting in highly purified cellulose. concentration of N-WS increased to 56.6%. After bleaching treatment, the cellulose content of B-WS increased to 87.9% due to the removal of remaining lignin and hemicelluloses, resulting in highly purified cellulose.

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hemicelluloses contents of N-WS decreased to 30.98% and 7.6%, respectively, whereas the cellulose

## *3.2. SEM Analysis*

The WS, N-WS, B-WS, TNC, and SNC microstructures were observed using a scanning electron microscope. The SEM image of WS (Figure 2a) denotes that the WS had a rough surface. However, the surface of N-WS (as seen in Figure 2b) had an irregular porous structure due to the degradation of hemicellulose and the partial degradation of lignin by the repeated alkali treatment. Further, B-WS (shown in Figure 2c) exhibited a loose structure, which indicated the successful removal of residual lignin by the acetate buffer/NaClO2 solution. These results are consistent with the changes in chemical composition denoted in Table 1. *3.2. SEM Analysis* The WS, N-WS, B-WS, TNC, and SNC microstructures were observed using a scanning electron microscope. The SEM image of WS (Figure 2a) denotes that the WS had a rough surface. However, the surface of N-WS (as seen in Figure 2b) had an irregular porous structure due to the degradation of hemicellulose and the partial degradation of lignin by the repeated alkali treatment. Further, B-WS (shown in Figure 2c) exhibited a loose structure, which indicated the successful removal of residual lignin by the acetate buffer/NaClO2 solution. These results are consistent with the changes in chemical composition denoted in Table 1.

**Figure 2.** SEM micrographs of (**a**) WS, (**b**) N-WS, (**c**) B-WS, (**d**) nanocellulose obtained via TEMPO oxidation (TNC), and (**e**) nanocellulose obtained via sulfuric acid hydrolysis (SNC). **Figure 2.** SEM micrographs of (**a**) WS, (**b**) N-WS, (**c**) B-WS, (**d**) nanocellulose obtained via TEMPO oxidation (TNC), and (**e**) nanocellulose obtained via sulfuric acid hydrolysis (SNC).

After the TNC was freeze-dried, the obtained aerogel was observed to be a porous network with a lamellar structure (Figure 2d). This structure is because the strong hydrogen bonding during freeze-drying caused the nanoparticles to self-assemble into the lamellar structures [53,54]. The aerogel formed by freeze-drying the SNC exhibited a similar porous structure (Figure 2e). After the TNC was freeze-dried, the obtained aerogel was observed to be a porous network with a lamellar structure (Figure 2d). This structure is because the strong hydrogen bonding during freeze-drying caused the nanoparticles to self-assemble into the lamellar structures [53,54]. The aerogel formed by freeze-drying the SNC exhibited a similar porous structure (Figure 2e).

#### *3.3. TEM Analysis 3.3. TEM Analysis*

TEM was used to observe the morphology of the nanocellulose produced using different methods. The prepared TNC (Figure 3a) exhibited an irregular block structure, whereas the prepared SNC (Figure 3b) was rectangular with a length of 55–82 nm and a width of 49–81 nm. The TNC morphology observed in this study was significantly different from that of the nanocellulose produced using the same procedure in a previous study [36,54]; this may be attributed to the irregular morphological structure [30] of the WS powder used in this study, as depicted in Figure 2a. Furthermore, the SNC morphology obtained through sulfuric acid hydrolysis as observed by TEM differed from the typical rod- or needle-like morphologies of the typical cellulose nanocrystals [2]. As has been reported, the size and shape of nanocelluloses influence the properties (for example, optical characteristics, stability, and rheology) in aqueous media [55], which largely determines the application of nanocellulose. Nanocellulose with spherical or square structure makes them excellent candidates as stabilizer for Pickering emulsion [53] or drug delivery carrier for encapsulation [56]. TEM was used to observe the morphology of the nanocellulose produced using different methods. The prepared TNC (Figure 3a) exhibited an irregular block structure, whereas the prepared SNC (Figure 3b) was rectangular with a length of 55–82 nm and a width of 49–81 nm. The TNC morphology observed in this study was significantly different from that of the nanocellulose produced using the same procedure in a previous study [36,54]; this may be attributed to the irregular morphological structure [30] of the WS powder used in this study, as depicted in Figure 2a. Furthermore, the SNC morphology obtained through sulfuric acid hydrolysis as observed by TEM differed from the typical rod- or needle-like morphologies of the typical cellulose nanocrystals [2]. As has been reported, the size and shape of nanocelluloses influence the properties (for example, optical characteristics, stability, and rheology) in aqueous media [55], which largely determines the application of nanocellulose. Nanocellulose with spherical or square structure makes them excellent candidates as stabilizer for Pickering emulsion [53] or drug delivery carrier for encapsulation [56].

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**Figure 3.** TEM images of (**a**) TNC and (**b**) SNC. **Figure 3.** TEM images of (**a**) TNC and (**b**) SNC.

#### *3.4. Chemical Structures 3.4. Chemical Structures*

modified by the TEMPO oxidation [63].

The changes in the chemical structures of the raw WS after various treatments were investigated using FTIR (Figure 4). Two main absorption regions appeared in all the curves among which one was in the high-wave-number region from 2800 to 3500 cm−1 and the other was in the low-wave-number region from 600 to 1750 cm−1 [57]. The peak at 2897 cm−1 was attributed to the stretching vibration of the C–H groups of cellulose, whereas the wide region around 3350 cm−1 was attributed to the O–H stretching vibration of the hydrogen-bonded hydroxyl groups in the cellulose molecules [51,58]. Further, the peak at 1738 cm−1 corresponds to the acetyl groups and ironic esters of the hemicellulose and the ester linkages of the carboxylic groups of the ferulic and p-coumaric acid in lignin and hemicellulose [59]. The absence of a peak at 1738 cm−1 in the FTIR spectrum of N-WS indicates that the hemicellulose in WS was effectively degraded by the NaOH treatment. The peaks at 1248 cm−1 and 1502 cm−1 in the FTIR spectrum of WS, corresponding to the aromatic skeletal vibrations of lignin [60], disappeared in the curve of B-WS, confirming that majority of the lignin was removed by the bleaching treatment. Furthermore, the absence of peaks at 1738 cm−1 , 1502 cm−1 , and 1248 cm−1 in the B-WS spectrum is consistent with the changes in chemical components during the alkaline and bleaching processes. The absorption peaks at 1162 cm−1 and 1034 cm−1 in all the curves correspond to the stretching vibration of the C–O–C bonds in the 1,4-glycosidic links linkages of the D-glucose units in cellulose, which were interpreted as typical for a cellulose structure [61]. The peaks at approximately 1645 cm−1 in the spectra of all the samples were attributed to the H–O–H stretching vibration of the adsorbed water due to the hydroxyl groups in cellulose [20], whereas those at 892 cm−1 represented the C1–H deformation of cellulose [62]. There were no significant differences between the SNC and B-WS spectra, demonstrating that the characteristics of the cellulose molecular structure were maintained during sulfuric acid hydrolysis. The increase in the relative amount of cellulose in the sample due to the decrease in the amounts of other components upon hydrolysis may account for the slight increase in the intensity of the peak at 1034 cm−1 from B-WS to SNC. The peak at 1738 cm−1 , which is characteristic of the C=O stretching of carboxyl groups, reappeared in the FTIR spectrum of TNC when compared with that of B-WS due to the introduction of –COOH on the cellulose surface; this indicates that cellulose was successfully The changes in the chemical structures of the raw WS after various treatments were investigated using FTIR (Figure 4). Two main absorption regions appeared in all the curves among which one was in the high-wave-number region from 2800 to 3500 cm−1 and the other was in the low-wave-number region from 600 to 1750 cm−1 [57]. The peak at 2897 cm−1 was attributed to the stretching vibration of the C–H groups of cellulose, whereas the wide region around 3350 cm−1 was attributed to the O–H stretching vibration of the hydrogen-bonded hydroxyl groups in the cellulose molecules [51,58]. Further, the peak at 1738 cm−1 corresponds to the acetyl groups and ironic esters of the hemicellulose and the ester linkages of the carboxylic groups of the ferulic and p-coumaric acid in lignin and hemicellulose [59]. The absence of a peak at 1738 cm−1 in the FTIR spectrum of N-WS indicates that the hemicellulose in WS was effectively degraded by the NaOH treatment. The peaks at 1248 cm−1 and 1502 cm−1 in the FTIR spectrum of WS, corresponding to the aromatic skeletal vibrations of lignin [60], disappeared in the curve of B-WS, confirming that majority of the lignin was removed by the bleaching treatment. Furthermore, the absence of peaks at 1738 cm−1 , 1502 cm−1 , and 1248 cm−1 in the B-WS spectrum is consistent with the changes in chemical components during the alkaline and bleaching processes. The absorption peaks at 1162 cm−1 and 1034 cm−1 in all the curves correspond to the stretching vibration of the C–O–C bonds in the 1,4-glycosidic links linkages of the d-glucose units in cellulose, which were interpreted as typical for a cellulose structure [61]. The peaks at approximately 1645 cm−1 in the spectra of all the samples were attributed to the H–O–H stretching vibration of the adsorbed water due to the hydroxyl groups in cellulose [20], whereas those at 892 cm−1 represented the C1–H deformation of cellulose [62]. There were no significant differences between the SNC and B-WS spectra, demonstrating that the characteristics of the cellulose molecular structure were maintained during sulfuric acid hydrolysis. The increase in the relative amount of cellulose in the sample due to the decrease in the amounts of other components upon hydrolysis may account for the slight increase in the intensity of the peak at 1034 cm−1 from B-WS to SNC. The peak at 1738 cm−1 , which is characteristic of the C=O stretching of carboxyl groups, reappeared in the FTIR spectrum of TNC when compared with that of B-WS due to the introduction of –COOH on the cellulose surface; this indicates that cellulose was successfully modified by the TEMPO oxidation [63].

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**Figure 4.** FTIR spectra of WS, N-WS, B-WS, TNC, and SNC. **Figure 4.** FTIR spectra of WS, N-WS, B-WS, TNC, and SNC. **Figure 4.** FTIR spectra of WS, N-WS, B-WS, TNC, and SNC.

#### *3.5. Crystal Structures 3.5. Crystal Structures 3.5. Crystal Structures*

The XRD patterns of WS, N-WS, B-WS, TNC, and SNC (Figure 5) were studied to further evaluate the influence of the processing treatment. All the samples exhibited diffraction peaks at approximately 16° (110) and 22.6° (200), whereas WS, N-WS, and B-WS also exhibited a diffraction peak at approximately 34° (004). These peaks indicate that the typical cellulose crystal structure was preserved in all the samples, indicating that the chemical and ultrasonic treatments did not change the integrity of the original cellulose crystal [64,65]. However, the crystallinity index changed with each step. The apparent crystallinity of WS was 29.5%. After NaOH treatment, the CrI of N-WS increased to 40.9% due to the dissolution and removal of lignin and majority of the hemicellulose (i.e., the amorphous hemicellulose) [57]. The CrI further increased to 42.9% as a result of the lignin removal by the acetate buffer/NaClO2 solution. These results are consistent with the observed changes in the chemical composition and the FTIR analyses. The CrI of SNC (40.1%) decreased slightly after the sulfuric acid hydrolysis and ultrasonication, which may have been caused because the amorphous and crystalline regions were damaged by the strong acid hydrolysis [66]. There were no significant differences in the position of X-ray diffraction peaks between the TNC and B-WS XRD curves, indicating that the original crystal structure of the cellulose was unchanged after the oxidation (Figure 5). These results indicate that the carboxylate groups formed by the TEMPO-mediated oxidation are selectively introduced on the surfaces of the cellulose microfibril rather than the internal cellulose crystallites [38]. The XRD patterns of WS, N-WS, B-WS, TNC, and SNC (Figure 5) were studied to further evaluate the influence of the processing treatment. All the samples exhibited diffraction peaks at approximately 16◦ (110) and 22.6◦ (200), whereas WS, N-WS, and B-WS also exhibited a diffraction peak at approximately 34◦ (004). These peaks indicate that the typical cellulose crystal structure was preserved in all the samples, indicating that the chemical and ultrasonic treatments did not change the integrity of the original cellulose crystal [64,65]. However, the crystallinity index changed with each step. The apparent crystallinity of WS was 29.5%. After NaOH treatment, the CrI of N-WS increased to 40.9% due to the dissolution and removal of lignin and majority of the hemicellulose (i.e., the amorphous hemicellulose) [57]. The CrI further increased to 42.9% as a result of the lignin removal by the acetate buffer/NaClO2 solution. These results are consistent with the observed changes in the chemical composition and the FTIR analyses. The CrI of SNC (40.1%) decreased slightly after the sulfuric acid hydrolysis and ultrasonication, which may have been caused because the amorphous and crystalline regions were damaged by the strong acid hydrolysis [66]. There were no significant differences in the position of X-ray diffraction peaks between the TNC and B-WS XRD curves, indicating that the original crystal structure of the cellulose was unchanged after the oxidation (Figure 5). These results indicate that the carboxylate groups formed by the TEMPO-mediated oxidation are selectively introduced on the surfaces of the cellulose microfibril rather than the internal cellulose crystallites [38]. The XRD patterns of WS, N-WS, B-WS, TNC, and SNC (Figure 5) were studied to further evaluate the influence of the processing treatment. All the samples exhibited diffraction peaks at approximately 16° (110) and 22.6° (200), whereas WS, N-WS, and B-WS also exhibited a diffraction peak at approximately 34° (004). These peaks indicate that the typical cellulose crystal structure was preserved in all the samples, indicating that the chemical and ultrasonic treatments did not change the integrity of the original cellulose crystal [64,65]. However, the crystallinity index changed with each step. The apparent crystallinity of WS was 29.5%. After NaOH treatment, the CrI of N-WS increased to 40.9% due to the dissolution and removal of lignin and majority of the hemicellulose (i.e., the amorphous hemicellulose) [57]. The CrI further increased to 42.9% as a result of the lignin removal by the acetate buffer/NaClO2 solution. These results are consistent with the observed changes in the chemical composition and the FTIR analyses. The CrI of SNC (40.1%) decreased slightly after the sulfuric acid hydrolysis and ultrasonication, which may have been caused because the amorphous and crystalline regions were damaged by the strong acid hydrolysis [66]. There were no significant differences in the position of X-ray diffraction peaks between the TNC and B-WS XRD curves, indicating that the original crystal structure of the cellulose was unchanged after the oxidation (Figure 5). These results indicate that the carboxylate groups formed by the TEMPO-mediated oxidation are selectively introduced on the surfaces of the cellulose microfibril rather than the internal cellulose crystallites [38].

**Figure 5.** XRD patterns of WS, N-WS, B-WS, TNC, and SNC. **Figure 5.** XRD patterns of WS, N-WS, B-WS, TNC, and SNC.

#### *3.6. Thermal Stability 3.6. Thermal Stability 3.6. Thermal Stability*

Figures 6 and 7 depicted the TGA and DTG curves, respectively, for WS, N-WS, B-WS, TNC, and SNC. All the TGA curves began with slight mass losses from room temperature to 105 ◦C, corresponding to the evaporation of slightly bound water from all the samples. As the temperature increased further, the WS degradation occurred in two phases. The first decomposition occurred between 214 ◦C and 300 ◦C, corresponding to the hemicellulose degradation and the beginning of lignin degradation [23,67]. After this change, the largest loss of mass in the material occurred between 300 ◦C and 380 ◦C, peaking at 342 ◦C, which corresponded to that of cellulose [68]. Figures 6 and 7 depicted the TGA and DTG curves, respectively, for WS, N-WS, B-WS, TNC, and SNC. All the TGA curves began with slight mass losses from room temperature to 105 °C, corresponding to the evaporation of slightly bound water from all the samples. As the temperature increased further, the WS degradation occurred in two phases. The first decomposition occurred between 214 °C and 300 °C, corresponding to the hemicellulose degradation and the beginning of lignin degradation [23,67]. After this change, the largest loss of mass in the material occurred between 300 °C and 380 °C, peaking at 342 °C, which corresponded to that of cellulose [68]. Figures 6 and 7 depicted the TGA and DTG curves, respectively, for WS, N-WS, B-WS, TNC, and SNC. All the TGA curves began with slight mass losses from room temperature to 105 °C, corresponding to the evaporation of slightly bound water from all the samples. As the temperature increased further, the WS degradation occurred in two phases. The first decomposition occurred between 214 °C and 300 °C, corresponding to the hemicellulose degradation and the beginning of lignin degradation [23,67]. After this change, the largest loss of mass in the material occurred between 300 °C and 380 °C, peaking at 342 °C, which corresponded to that of cellulose [68].

*Polymers* **2019**, *11*, x FOR PEER REVIEW 9 of 14

**Figure 6.** TGA curves of WS, N-WS, B-WS, TNC, and SNC. **Figure 6.** TGA curves of WS, N-WS, B-WS, TNC, and SNC. **Figure 6.** TGA curves of WS, N-WS, B-WS, TNC, and SNC.

**Figure 7.** DTG curves of WS, N-WS, B-WS, TNC, and SNC. **Figure 7.** DTG curves of WS, N-WS, B-WS, TNC, and SNC.

**Figure 7.** DTG curves of WS, N-WS, B-WS, TNC, and SNC. In contrast, the decomposition of four other samples occurred in only one stage. N-WS began to degrade at 205 °C due to lignin degradation; however, the maximum mass loss occurred at 301 °C, which can be attributed to cellulose degradation. The absence of peaks related to the degradation of In contrast, the decomposition of four other samples occurred in only one stage. N-WS began to degrade at 205 °C due to lignin degradation; however, the maximum mass loss occurred at 301 °C, which can be attributed to cellulose degradation. The absence of peaks related to the degradation of hemicellulose and the relatively small change associated with lignin degradation in this sample as In contrast, the decomposition of four other samples occurred in only one stage. N-WS began to degrade at 205 ◦C due to lignin degradation; however, the maximum mass loss occurred at 301 ◦C, which can be attributed to cellulose degradation. The absence of peaks related to the degradation of hemicellulose and the relatively small change associated with lignin degradation in this sample as

hemicellulose and the relatively small change associated with lignin degradation in this sample as

compared with that associated with WS indicates that the alkaline treatment eliminated most of the hemicellulose and some of the lignin from the WS.

B-WS degraded from 222 to 380 ◦C, with the most significant mass loss occurring at 332 ◦C; this was mainly attributed to cellulose degradation, which was within the decomposition temperature range reported in previous studies [68,69] (315–400◦C). The wide range, which was associated with lignin degradation, was not observed in this sample, indicating that lignin was eliminated after the bleaching reaction in the acetate buffer/NaClO2 solution.

The thermal degradation of TNC (i.e., following TEMPO/NaClO/NaBr oxidation) began at 178 ◦C, which was approximately 50 ◦C lower than that in B-WS. It has been reported that the introduction of –COOH by the TEMPO-mediated oxidation of the C6 primary hydroxyl groups on the cellulose surface results in a significant decrease in thermal degradation [70].

SNC exhibited the worst thermal stability among the five tested samples, which can be attributed to the introduction of sulfate groups. It had been reported that the degradation of sulfate groups requires relatively low activation energy [71,72]. The thermal stability of TNC was likely to be lower than that of SNC because it exhibited fewer crystalline regions. This result is consistent with those in previous studies [73,74], which have shown that the crystalline regions of nanocellulose generally provide thermal stability. This finding is in agreement with the finding of XRD analyses, which showed that TNC had a high crystallinity index.

#### **4. Conclusions**

In this study, nanocellulose was produced using WS as the raw material; novel shapes were obtained by applying different production processes. The chemical composition analysis revealed that the hemicellulose and lignin in WS were effectively removed by alkali treatment and bleaching, increasing the cellulose content to 89%. The TEM images denoted that the produced TNC exhibited an irregular block structure, whereas SNC was rectangular with a length of 55–82 nm and a width of 49–81 nm. The results of the FTIR and XRD analyses of WS, N-WS, and B-WS were consistent with the observed chemical changes and confirmed that the typical cellulose structure remained unchanged through the TEMPO oxidation and sulfuric acid hydrolysis treatments. Our observations demonstrate that WSs, which are an abundant and sustainable agricultural waste, may be repurposed for the production of nanocellulose.

**Author Contributions:** Conceptualization, D.Z.; Formal analysis, D.Z.; Investigation, D.Z.; Methodology, D.Z.; Project administration, J.Y.; Resources, J.Y.; Supervision, J.Y.; Validation, Y.Z. and Y.G.; Writing—original draft, D.Z.; Writing—review & editing, D.Z. and J.Y.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors express their appreciation to Key Laboratory of Biobased Material Science and Technology of Ministry of Education and College of Material Science and Engineering of Northeast Forestry University for full support of this research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

1. Harini, K.; Ramya, K.; Sukumar, M. Extraction of nano cellulose fibers from the banana peel and bract for production of acetyl and lauroyl cellulose. *Carbohydr. Polym.* **2018**, *201*, 329–339. [CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **E**ff**ect of Cellulose Nanocrystals from Di**ff**erent Lignocellulosic Residues to Chitosan**/**Glycerol Films**

**Marina Reis de Andrade 1 , Tatiana Barreto Rocha Nery 2 , Taynã Isis de Santana e Santana 1 , Ingrid Lessa Leal 2 , Letícia Alencar Pereira Rodrigues 2 , João Henrique de Oliveira Reis 3 , Janice Izabel Druzian 3 and Bruna Aparecida Souza Machado 2,4,\***

Received: 11 March 2019; Accepted: 8 April 2019; Published: 10 April 2019

**Abstract:** Interest in nanocellulose obtained from natural resources has grown, mainly due to the characteristics that these materials provide when incorporated in biodegradable films as an alternative for the improvement of the properties of nanocomposites. The main purpose of this work was to investigate the effect of the incorporation of nanocellulose obtained from different fibers (corncob, corn husk, coconut shell, and wheat bran) into the chitosan/glycerol films. The nanocellulose were obtained through acid hydrolysis. The properties of the different nanobiocomposites were comparatively evaluated, including their barrier and mechanical properties. The nanocrystals obtained for coconut shell (CS), corn husk (CH), and corncob (CC) presented a length/diameter ratio of 40.18, 40.86, and 32.19, respectively. Wheat bran (WB) was not considered an interesting source of nanocrystals, which may be justified due to the low percentage of cellulose. Significant differences were observed in the properties of the films studied. The water activity varied from 0.601 (WB Film) to 0.658 (CH Film) and the moisture content from 15.13 (CS Film) to 20.86 (WB Film). The highest values for tensile strength were presented for CC (11.43 MPa) and CS (11.38 MPa) films, and this propriety was significantly increased by nanocellulose addition. The results showed that the source of the nanocrystal determined the properties of the chitosan/glycerol films.

**Keywords:** films; nanocellulose; nanocrystals; biodegradable packaging

## **1. Introduction**

With the advancement of nanotechnology and nanoscience, materials are modulated in their technologies, generating new technologies to incorporate to the needs of the current society [1–5]. In this context, cellulose nanocrystals are being used to improve the mechanical and barrier properties of chitosan films and make their commercialization viable [6–11]. Nanocellulose or cellulose nanocrystals are the crystalline domains of cellulosic sources, obtained through acid hydrolysis, having characteristics of high rigidity, high crystallinity, and nanometric size [6–10]. The cellulose nanocrystals have been the object of several studies, since they present great potential of application as reinforcement in polymer matrices [12,13]. Cellulose nanocrystal is known as the most appropriate and efficient reinforcement additive due to its renewability, excellent mechanical properties, and economic cost [14], and can be obtained from waste, further improving the cost benefit.

Among the nanoreinforced materials, the application of the vegetal fibers stands out, since these have ample availability in almost all the countries, being usually designated as lignocellulosic materials [15–17]. Some fibers are found spontaneously in nature, while others are derived from agricultural activity and from waste generated mainly by agroindustry. Studies evaluate the application of nanocrystals obtained by several fibers in polymeric systems, for example, barley straw and husk in poly(vinyl alcohol) (PVA) blended with natural chitosan (CH) nanocomposites [18], pine cones in a biodegradable poly(3-hydroxybutyrate)/poly(ε-caprolactone) (PHB/PCL) [19], and sunflower stalks on wheat gluten bionanocomposites [20]. The high availability of lignocellulosic fibers, coupled with the need for a renewable source for the production of polymers, represents a great opportunity for technological advances that add value to the products or residues of the agroindustry and, at the same time, act in the fixation of carbon in nature [21–24]. This implies helping to reduce the emission of CO2 into the atmosphere during the production cycle, increasing the economic potential of agribusiness due to the possibility of trading carbon credits in the production chain [25–29].

In relation to the development of new materials, there is also the growth of technologies using polymers from renewable sources for diverse applications. These materials have been important for the advancement of the sciences, and have several advantages such as being easily obtainable, biocompatible, and biodegradable [30,31]. The choice of material to be used in the formulation of the films is very important, as they will depend on the interactions between the components of the material, which may interfere with the barrier properties, mechanical properties, and the physical aspects of the films [32].

Several biopolymers such as polysaccharides, proteins, and lipids have been used as polymer matrices for the development of biodegradable packaging due to their availability, renewability, low cost, respect for the environment, and biodegradability. Among these, chitosan is considered favorable for the development of biocomposites [33,34], and based on production volumes, is the second most abundant polymer after cellulose [35,36]. Chitosan is a natural polysaccharide derived from chitin, and although the most important sources are commercial shellfish carapaces, studies indicate that this element can be found in insects, mollusks, and fungal cell walls [37]. The bioactivity of this material has aroused interest in the application as a packaging film due to its ability to form flexible and resistant films with efficient oxygen barrier and antimicrobial activity [6,7,9,38,39]. In addition, the use of chitosan in the field of biomedicine has been reported for its various important pharmacological properties and its role in tissue engineering, regenerative medicine, scaffold, and drug delivery systems is also well documented [40].

Chitosan based films are biocompatible and biodegradable, with excellent mechanical strength and cost effectiveness [41]. Previous studies have shown the efficiency of the incorporation of nanocrystals obtained from different sources into chitosan films, contributing to increase the mechanical and barrier properties of these materials [42–44]. Azeredo et al. [45] evaluated the effect of different concentrations of nanocellulose on tensile properties and water vapor permeability of chitosan films. Pereda et al. [46] demonstrated that the combined use of cellulose nanoparticles and olive oil proved to be an efficient method to reduce the inherently high water vapor permeability of plasticized chitosan films, improving their tensile behavior at the same time.

The films obtained by natural polymers are poorly flexible and brittle, thus, it becomes necessary to add plasticizer to the polymer matrix to improve its flexibility characteristics. Plasticizers reduce the interactions between adjacent molecules, increasing film flexibility [47]. For application of a plasticizer, it is extremely important that it is suitably compatible with the polymer used and the definition of proportionality between the components in order to tailor the final composition to a given application [48]. Several plasticizers are used in the preparation of biodegradable films and coatings, including mono-, di- and oligosaccharides (glucose, sucrose); polyols (glycerol, sorbitol, derivatives of glycerol); and lipids (saturated fatty acids, monoglycerides and ester derivatives, phospholipids and

surfactants) [49]. The use of plasticizers in these films allows a greater percentage of elongation and adaptation of the matrix in the structure [50]. Glycerol is currently one of the plasticizers most used in the development of biodegradable films, which has caused scientific impact since this polyalcohol is a byproduct generated from the biodiesel chain, which is expanding worldwide [51], and is thus a low-cost and high-availability material.

With this background, the objective of this work was the extraction of cellulose nanocrystals from four different lignocellulosic fibers, considered as byproducts of the agro-food industries (Corncob (CC), corn husk (CH), wheat bran (WB) and coconut shell (CS)) and the investigation of the influence of the incorporation of these nanoparticles on the physical, barrier, and mechanical properties in chitosan biofilms. The main focus of the study is to identify sources of residues for the production of cellulose nanocrystals and, consequently, to evaluate the behavior of the inclusion of these nanoparticles in films using chitosan as a polymer matrix. Chitosan is identified as a highly attractive biomaterial for film owing to its properties seen in previous reports. Chitosan can be easily incorporated into gels, membranes, beads, and scaffolds, and these forms provide a wide variety of biomedical applications and food packaging.

### **2. Materials and Methods**

For extraction of the cellulose nanocrystals were used corncob (CC) and corn husk (CH) bought in local commerce in Salvador, Bahia, Brazil, wheat bran (WB) provided by a local wheat mill, and coconut shell (CS) donated by Frisbraztech (Conde, Bahia, Brazil). The films were produced with chitosan (Sigma-Aldrich, Saint Louis, MO, USA, Cas Number: 9012-76-4, with a degree of deacetylation ≥ 75%), and the glycerol purchased from Synth (São Paulo, Brazil).

#### *2.1. Characterization of the Fibers*

The natural fibers were characterized as moisture, water activity, ash content, and crude fiber content.

The moisture content was determined using an infrared-heated scale (Shimadzu, MOC-120H, Kyoto, Japan) with the intensity of the emitted radiation set so that the sample would reach 105 ◦C. Measurements of water activity were performed using a Decagon (Novasina®, Lab Master aw, Neuheimstrasse, Switzerland) at temperature of 25 ◦C. The ash content was determined using an muffle Fornitec, and crude fiber content (lignin, hemicellulose and cellulose) in Ankom A200 Fiber Analyzer (New York, NY, USA), by the FDA (Acid Detergent Fiber) and NDF (Neutral Detergent Fiber), according to the methodology proposed by Van-Soest, Robertson and Lewis [52].

### *2.2. Extraction of Cellulose from Fibers*

The extraction of the cellulose pulp was performed based on the works of Samir et al. [53] and Machado et al. [12]. The selected materials (CC, CH, WB and CS) were previously dried at 60 ◦C for 3 h to remove excess moisture and ground in a blender to obtain a fine powder. The crushed fibers (30 g) were washed in 1200 mL of 2% NaOH solution and at 80 ◦C for a period of 4 h under constant stirring. The resulting solutions were filtered and washed with water to obtain a pulp. The washing process was repeated three times for complete removal of the water soluble agents and obtaining the cellulose pulp. After washing, the process of delignification and bleaching of pulps was carried out, using 300 mL of sodium hypochlorite (1.7%) and 300 mL of a buffer solution. The resulting solution was placed under constant stirring at a temperature of 80 ◦C for 6 h (TE-394/2, Tecnal, São Paulo, Brazil), filtered and oven dried (40 ◦C) to obtain the cellulose pulp of each fiber.

#### *2.3. Preparation of Cellulose Nanocrystals*

The cellulose nanocrystals were prepared by acid hydrolysis using 45% H2SO4 [12,38]. Briefly, 12 mL/g of cellulose was subjected to constant stirring for period of 1 h, and temperature between 50 and 55 ◦C. After acid hydrolysis, the dispersions were cooled to 30 ◦C and the volume was completed

to 40 mL in Falcon tubes. The tubes were centrifuged for 10 minutes at 4400 rpm and a temperature of 10 ◦C (Sigma 2-16KHL, Osterode am Harz, Germany) in order to separate the crystals in suspension. Centrifugation was repeated 4 to 6 times for better separation results.

Then the suspensions were subjected to dialysis using cellulose membranes (D9777-100 FTO, 12.000 Da cut off, Sigma-Aldrich, Saint Louis, MO, USA), and after reaching the pH between 6 to 7, the samples were placed in an ultrasonic bath (RMS, Quimis, São Paulo, Brazil) with a power of 200 W, frequency of 60 kHz, and a temperature of 25◦C for 5 min for the dispersion of the nanocrystals.

#### *2.4. Production of Films (Nanobiocomposites)*

The films with nanoparticles extracted from different fibers were produced by the casting method, according to the methodology proposed by Yassue–Cordeiro et al. [31] and de Souza [54]. Chitosan (1.5%, g/100g) dissolved in glacial acetic acid (1.0%, g/100g) and glycerol (0.15%, g/100g) was used as the plasticizing agent. To this mixture was added 5% (w/v) cellulose nanocrystals (a filmogenic solution for each fiber) under constant shaking in Shaker Incubator (MA420, Marconi, São Paulo, Brazil) at 40 ◦C for 24 h (Table 1). After homogenization, 40 g of the solution was transferred to Petri dishes and subjected to dehydration in an air circulation oven (35 ± 2 ◦C) (Q314M, Quimis, São Paulo, Brazil) for 20 h. Other film was produced without the addition of cellulose nanocrystals for use as control. The films obtained were packed in a vacuum desiccator containing saturated sodium chloride solution (TE-3950, Tecnal, São Paulo, Brazil).

**Table 1.** Formulation of films containing nanocrystals of different lignocellulosic sources and control.

Coconut shell (CS), corn husk (CH), corncob (CC) and wheat bran (WB).

#### *2.5. Characterization of Cellulose Nanocrystals*

The concentration of nanocrystalline cellulose in the suspensions was determined by gravimetric analysis. An aliquot with a known volume was dried at 40 ◦C for 24 h in an air circulation oven (TE-394/2, Tecnal, São Paulo, Brazil). The birefringence of the nanocellulose suspensions was determined by the methodology proposed by Flauzino et al. [55], an aliquot of aqueous suspension of the nanocrystals (5 x 10−3 g.mL−1 ) was placed in a glass test tube, this tube was placed in front of a polarized light source, and it was then photographed with a camera equipped with a filter of polarized light. Cellulose nanocrystalline dispersions for all fibers were analyzed by transmission electron microscopy (TEM), in order to determine the length of the fibers (L), width (D), and aspect ratio (L/D) and indicate the state of aggregation of the crystals. Measurements were made directly from the micrographs using Image Tool 6.3 (Media Cybernetics, Rockville, USA) with 30 measurements to determine mean and standard deviation values according to the methodology proposed by Machado et al. [12].

#### *2.6. Characterization of Films—Determination of Thickness and Mechanical Properties*

The thickness of the films and control were evaluated in random positions using digital micrometer (Digimess, São Paulo, Brazil) with resolution of 0.001 mm. The tensile tests were performed using a Brookfield (Braseq CT310K, Middleboro, MA, USA), with a maximum load of 10 KN, with a speed of 0.5 mm s−1 , a temperature of 25 ◦C, trigger load of 7 g, test probe tip of TA3/100 and TA / TPB device [13,56]. Tensile tests were performed on six specimens with dimensions of 80 mm in length and 25 mm wide for each sample.

## *2.7. Statistical Analysis*

The results of this study were expressed as the mean±standard deviation (sd) (n = 3). The statistical analysis of the results was performed using the Statistica® 6.0 software from StatSoft (Tulsa, Hamburg, Germany). The results were treated by the Tukey test to identify if the changes in the parameters evaluated were significant at the 95% level of significance.

#### **3. Results**

## *3.1. Characterization of Fibers*

The composition and structure of lignocellulosic biomass have great influence on the nature and yields of the hydrolysis processes. The moisture content (%), the water activity (aw), and the ash content (%) of the fibers obtained from corn cob (CC), corn husk (CH), wheat bran (WB), and coconut shell (CS) are shown in Table 2.

**Table 2.** Mean values of moisture content, water activity, and ash of lignocellulosic fibers (mean ± standard deviation).

Coconut shell (CS), corn husk (CH), corncob (CC), and wheat bran (WB). Mean ± standard deviation of samples. Values with the same letter in the same column did not present significant differences (*p* < 0.05) by Tukey's test at 95% confidence (a–d).

Table 3 shows the cellulose, hemicellulose and lignin contents of the fibers studied. Each component of the lignocellulosic fibers is responsible for different functions, so different levels of these components influence the properties of the nanocellulose fibers obtained [38,57,58].

**Table 3.** Cellulose, hemicellulose, and lignin contents in natural lignocellulosic fibers (mean ± standard deviation).

Coconut shell (CS), corn husk (CH), Corncob (CC), and wheat bran (WB). Mean ± standard deviation of samples. Values with the same letter in the same column did not present significant differences (*p* < 0.05) by Tukey's test at 95% confidence (a-d).

### *3.2. Characterization of Cellulose Nanocrystals*

Figure 1 shows the four steps of washing the fiber with NaOH for the process of extracting cellulose from the corncob, as an example, until the bleaching stage.

For each 30 g of fiber submitted to the washing and bleaching process (Figure 1), different yields of cellulose pulp were obtained, and these results are presented in Table 4. The concentration of nanocrystals (g.10mL−1 ) is a parameter for the evaluation of the nanocellulose dispersion, indicating if it needs a higher concentration for use in the production of the films.

The existence of nanocrystals can be proven by birefringence analysis and microscopy. According to Pereira et al. [59], flow birefringence results from the alignment of nanoparticles and indicates the existence of isolated nanocrystals in the dispersion. As the cellulose nanocrystals are rigid rod-like particles, they have a strong tendency to align a vector director and increase in relation to particle size/diameter. As a result of the strong birefringence of the native cellulose, this rod alignment creates a macroscopic

sources.

birefringence that can be directly observed though cross polarizers [60]. The suspensions (CH, CC, CS and WB) were analyzed from a polarizing lens. In the birefringence analysis, the CC nanocrystals dispersion was the one that presented the nematic phase liquid crystals (N) clearly to prove the existence of the crystals (Figure 2), CH and CS presented nematic phase with less clarity, and the WB did not present *Polymers*  nematic phase. This information is in agreement with the results presented in Table **2018** 5. , *10*, x FOR PEER REVIEW 6 of 16

**Figure 1.** Cellulose pulp obtained by corncob (CC): (**a**) first wash with NaOH; (**b**) second washing; (**c**) third washing; (**d**) fourth washing; (**e**) bleaching step. **Figure 1.** Cellulose pulp obtained by corncob (CC): (**a**) first wash with NaOH; (**b**) second washing; (**c**) third washing; (**d**) fourth washing; (**e**) bleaching step.

For each 30 g of fiber submitted to the washing and bleaching process (Figure 1), different yields **Table 4.** Yield and concentration of cellulose pulp and nanocrystals in the different lignocellulosic sources.

**Table 4.** Yield and concentration of cellulose pulp and nanocrystals in the different lignocellulosic *Polymers* **2018**, *10*, x FOR PEER REVIEW Coconut shell (CS), corn husk (CH), corncob (CC), and wheat bran (WB).

7 of 16

alignment creates a macroscopic birefringence that can be directly observed though cross polarizers [60]. The suspensions (CH, CC, CS and WB) were analyzed from a polarizing lens. In the birefringence analysis, the CC nanocrystals dispersion was the one that presented the nematic phase **Figure 2.** Phenomenon of birefringence observed through a polarized lens after dispersion of cellulose nanocrystals extracted from corncob (CC). **Figure 2.** Phenomenon of birefringence observed through a polarized lens after dispersion of cellulose nanocrystals extracted from corncob (CC).

liquid crystals (N) clearly to prove the existence of the crystals (Figure 2), CH and CS presented Table 5 presents the mean values of the width (D), length (L), and the L/D ratio of the crystals. **Table 5.** Size of the nanocrystals of different lignocellulosic sources (mean ± standard deviation).

WB - - - Coconut shell (CS), corn husk (CH), corncob (CC), and wheat bran (WB). L = length; D = width and L / D ratio. Coconut shell (CS), corn husk (CH), corncob (CC), and wheat bran (WB). L = length; D = width and L/D ratio.

CC, CH, and CS presented positive results for TEM analysis, where it was possible to visualize the aggregate crystals in a needle format (Figure 3). It was not possible to visualize crystal formation

and acid-wood pulp ratio on the properties and behavior of the whisker suspension, obtained by sulfuric acid hydrolysis, was observed that shorter whiskers, less variable in length, were obtained in longer reaction times [61]. Beck-Candanedo et al. [61] also found out that an increase in the acid-

for wheat fiber, indicating that they are not present in a considerable amount.

pulp ratio also leads to whiskers with reduced dimensions.

Table 5 presents the mean values of the width (D), length (L), and the L/D ratio of the crystals.

CC, CH, and CS presented positive results for TEM analysis, where it was possible to visualize the aggregate crystals in a needle format (Figure 3). It was not possible to visualize crystal formation for wheat fiber, indicating that they are not present in a considerable amount. *Polymers* **2018**, *10*, x FOR PEER REVIEW 8 of 16

**Figure 3.** Cellulose nanocrystals obtained by Transmission Electron Microscopy (TEM) (PTA contrast and uranyl). (**a**) and (**b**) Corn husks; (**c**) and (**d**) Corncob; (**e**) and (**f**) Wheat bran; (**g**) and (**h**) Coconut shell (Scale: 200 nm). **Figure 3.** Cellulose nanocrystals obtained by Transmission Electron Microscopy (TEM) (PTA contrast and uranyl). (**a**) and (**b**) Corn husks; (**c**) and (**d**) Corncob; (**e**) and (**f**) Wheat bran; (**g**) and (**h**) Coconut shell (Scale: 200 nm).

The process conditions, whether concerning fiber preparation or hydrolysis for whisker isolation, affect the morphological characteristics of these nanomaterials. The acid used for hydrolysis may affect the characteristics of whisker dispersion in an aqueous system. The effect of reaction time and acid-wood pulp ratio on the properties and behavior of the whisker suspension, obtained by sulfuric acid hydrolysis, was observed that shorter whiskers, less variable in length, were obtained in longer reaction times [61]. Beck-Candanedo et al. [61] also found out that an increase in the acid-pulp ratio also leads to whiskers with reduced dimensions.

### *3.3. Production and Characterization of Films*

physical appearance of CS film.

The films produced from the formulations described in Table 1, using different types of nanocrystals, as well as the control film, were analyzed to determine their physical and barrier properties (water activity (aw), moisture content (M), total solids (TS), and thickness (*t*)) and their mechanical properties (tensile strength (σ) and strain (ε)), described in Table 6. Figure 4 shows the physical appearance of CS film.

**Table 6.** Characterization of nanobiocomposites (mean ± standard deviation).

Coconut shell (CS), corn husk (CH), corncob (CC), and wheat bran (WB). Values followed by same letter, in the same column, did not present significant differences (*p* > 0.05) by the Tukey test at 95% confidence (a-d). mechanical properties (tensile strength (σ) and strain (ε)), described in Table 6. Figure 4 shows the

**Figure 4.** Physical appearance of CS film. **Figure 4.** Physical appearance of CS film.

**Table 6.** Characterization of nanobiocomposites (mean ± standard deviation).

#### **4. Discussion**

**4. Discussion**

**Film aw±sd M ± sd (%) TS ± sd (%) t ± sd (mm) σ ± sd (MPa) ε ± sd (%) Control** 0.610±0.01b 20.75±0.78a 78.92±0.78d 0.049±0.02a 4.08±1.87d 115.9±4.36e **CS Film** 0.600±0.01b 15.13±0.01c 84.87±0.01a 0.040±0.04ab 11.38±3.53a 274.2±1.35a **CH Film** 0.658±0.02a 20.24±0.62a 79.76±0.62c 0.027±0.01bc 6.99±4.56c 155.2±5.13c Among the fibers, CS had the highest results to moisture (88.7% ± 0.07), water activity (0.97 ± 0.05), and ash (5.37% ± 0.10), the WB presented the smaller moisture content (12.4% ± 0.60), and water activity (0.640 ± 0.03) (Table 2). The moisture content of the fibers, as well as the storage conditions and the time, can interfere in the degree of crystallinity of the cellulose, and consequently, in obtaining

by the wheat variety, the maturation stage of the samples, and the producer region.

Among the fibers, CS had the highest results to moisture (88.7% ± 0.07), water activity (0.97 ± 0.05), and ash (5.37% ± 0.10), the WB presented the smaller moisture content (12.4% ± 0.60), and water activity (0.640 ± 0.03) (Table 2). The moisture content of the fibers, as well as the storage conditions and the time, can interfere in the degree of crystallinity of the cellulose, and consequently, in obtaining the nanocrystals [15,29,54]. The CH and CC fibers analyzed in this study were collected from ears of green and not dry corn. For this reason, the values found for moisture and water activity were higher than those found in the literature. Corn husk presented higher moisture than that presented by Reference [62] (12.96%) and near ash content (1.52%), which can be explained mainly by the origin of the fiber and its level of maturation. Ziglio et al. [63] found moisture values of 8.9% for cob corn [63]. The results of moisture found for wheat bran are similar to those presented by De Lima Dantas et al. [64], 11.59%, however, the ash value was lower (0.62%). Differences can be justified

The results in Table 3 show the differences in fiber composition of the different residues studied. The CC fiber showed the highest values of cellulose (52.99% ± 1.79) followed by the CS fiber (47.16%

**CC Film** 0.611±0.05b 18.32±0.90b 81.68±0.90b 0.019±0.01c 11.43±3.58a 195.2±8.76b

the nanocrystals [15,29,54]. The CH and CC fibers analyzed in this study were collected from ears of green and not dry corn. For this reason, the values found for moisture and water activity were higher than those found in the literature. Corn husk presented higher moisture than that presented by Reference [62] (12.96%) and near ash content (1.52%), which can be explained mainly by the origin of the fiber and its level of maturation. Ziglio et al. [63] found moisture values of 8.9% for cob corn [63]. The results of moisture found for wheat bran are similar to those presented by De Lima Dantas et al. [64], 11.59%, however, the ash value was lower (0.62%). Differences can be justified by the wheat variety, the maturation stage of the samples, and the producer region.

The results in Table 3 show the differences in fiber composition of the different residues studied. The CC fiber showed the highest values of cellulose (52.99% ± 1.79) followed by the CS fiber (47.16% ± 1.24). The CC fiber presented the highest hemicellulose content while the CS fiber had the highest lignin content (30.71 ± 0.21). Souza et al. [65] found values of 37.6% cellulose, 34.5% hemicellulose, and 12.6% lignin for corn husk, and 31.7% cellulose, 34.7% hemicellulose, and 20.3% lignin for corn cob. In the literature, different values of cellulose (56.8%) and lignin (29.8%) were presented for coconut shell [66].

In their study, Merali et al. [67] found values of 18.5% cellulose, 54.8% hemicellulose, and 10.8 lignin in pretreated WB hydrothermally. These values may be associated to the exchange rate in the fiber, in the region where it was extracted and its botanical varieties. Mendes et al. [68] found values of 33–40% cellulose, 33–40% hemicellulose, and 2–16% lignin in CH residue samples. The values found by the authors show the large range of these constituents in the samples, which can be attributed to maize variety and harvest period.

The importance of determining cellulose at the fibers is that being a polysaccharide made of repeating beta 1,4-glycosidic bonds, it is characterized by having intercalated arrangements of highly ordered (crystalline) and amorphous (disordered) [69]. Thus, the isolation and obtaining of nanocrystals from lignocellulosic fibers depend directly on the proportion of these crystalline regions, as well as the lignin and hemicellulose content, since they interfere in the extraction process, the realization of pretreatment for removal of these components being necessary in many cases [28,31].

The visualization of the suspension of the cellulose nanocrystals obtained from the fibers studied using polarizers revealed a nematic phase, which was directly produced by light birefringence. This result was also important to confirm the presence of nanocrystals (except for WB) and is considered an important analysis to evaluate nanocrystals dispersion (Figure 2). Cerqueira et al. [13] and Alves et al. [17] similarly used crossed polarizers to visualize the birefringence phenomenon in suspension of cellulose nanocrystals obtained from coconut and eucalyptus, respectively. The nematic liquid crystal phase combines long-range orientational order with regular liquid-like short-range positional order. In a nematic CNC suspension, the nanocrystals align preferentially with their long axes along a common direction [70]. Perhaps there is a correlation among nematic phase (preferential orientation of the nanocrystals), refractive effects, and size of the CNC. In this study, a positive correlation was observed regarding the size of the nanocrystals obtained and the presence of birefringence. For example, the best nematic phase was presented by CC, which also has the highest L and D, and consequently, lower L/D ratio.

The morphological analysis is of great importance in determining the size and the state of agglomeration, considering that the source of cellulose as well as the technique used for the hydrolysis of the amorphous structure influence the size and the final properties of the nanoparticles [71,72]. Table 5 presents the mean values of the width (D), length (L), and the L/D ratio of the crystals. It is observed that the CC presented higher values of L and D and a lower L/D ratio. The values of (L) are in agreement with the literature [57,73] indicating a great potential of use of this fiber as reinforcement for bionanocomposites, as demonstrated in other studies. Similar results were reported by Machado et al. [74] (L = 98–430 nm, D = 6 nm e L/D = 38.9 ± 4.7), Rosa et al. [75] (L = 197 nm, D = 5.8 nm e L/D = 39), and Sarwar et al. [76] (240–280 nm).

Oliveira [77] notes that the resulting nanoparticle dimensions depend on the cellulose source and the hydrolysis process. Smaller nanoparticle diameters may be associated with higher amounts of hemicellulose present in the fiber structure, which would limit the organization of cellulose chains [78,79]. In this study, the CC fiber presented the highest concentration of cellulose and, consequently, presented the lowest L/D ratio for the nanocrystals obtained. The L/D ratio ranged from 32.19 to 40.86, being in agreement with other studies that presented values between 18.2 and 75.4 nm [13,79]. Some authors [11,80,81] found similar dimensions for the coconut shell. In this range, the crystals have great potential to be used as reinforcement in biodegradable films.

The film made with CH cellulose nanocrystals had a higher value of water activity (0.658), while the others presented approximate values of 0.600. The reduction of free water in packages for food products has the consequence of reducing the growth of microorganisms, avoiding undesirable chemical changes in the storage of the products [82–85]. Products that have water activity values lower than 0.600 are relatively protected against microbial contamination, whereas the proliferation of specific microorganisms can occur with water activity values above 0.600 [86–88]. Associated with the amount of free water of the product, the moisture of the film, which favors or inhibits proportionally the proliferation of microorganisms, is also evaluated [7,89].

The films produced with cellulose nanocrystals of CC and CS presented lower moisture content (18.32 ± 0.90 and 15.13 ± 0.01) in relation to films produced with WB and CH nanocrystals (20.86 and 20.24). In relation to the total solids, an increase is observed for the CC and CS, indicating that the amount of nonvolatile or water insoluble particles is slightly higher.

The thickness of the films (Table 6) was lower than the control film, maintaining unchanged barrier properties. Because it is a manual process, the standardization of the fluid distribution and the drying process are difficult. In a study, Hänninen et al. [90], observed that by adding a birch cellulose nanofiber, the thickness doubled compared to the control film containing only chitosan.

For this work, it was also observed that the incorporation of the nanocellulose dispersion, from the various fibers, to the film plasticized with glycerol, resulted in the improvement of the mechanical properties of the formulations studied. The tensile strength values (σ) varied between 4.08 MPa (control) to 11.38 MPa (CS Film) and 11.43 MPa (CC Film), presenting significant differences among the samples evaluated (except to CS and CC). The biofilms with the CS and CC nanocrystals dispersion presented a higher tensile strength at the rupture, indicating that a greater force was required for the rupture of the films, suggesting that there was an increase of the resistance. Regarding the deformation, the values found ranged from 115.9% (control) to 274.2% (CS film), and the WB film (110.0% ± 90.9) had a lower percentage of the evaluated fibers, with values close to the control. The lowest results in relation to the mechanical properties for the WB film may be justified due to the lowest concentration of cellulose in the fiber, and consequently the lowest efficiency to obtain the nanocrystals. This was also confirmed because of the impossibility of determining the size of the nanocrystals obtained by TEM (due to low concentration and formation of few clusters) as well as, no nematic phase formation. Benini (2011) [91] incorporated high impact polystyrene (HIPS) coconut fibers as a thermoplastic matrix, and the maximum stress and Young modulus were 23.7 MPa and 3.0 MPa for composites containing 30% of fibers.

The effect of surface and dispersion characteristics of the whiskers used as reinforcement material in a matrix with polypropylene was investigated by Ljungberg et al. [92]. These authors observed that the quality of nanocrystal dispersion is an important aspect, which affects the quality of the film, making the films more opaque and influencing film strength.

It is possible to visualize an increase in the mechanical resistance of the films with the incorporation of the nanocrystals dispersion, making them more rigid, but there was an increase in the percentage of deformation for all the films (Table 6). The stiffness in some materials can negatively interfere in the percentage of deformation of the same, the more rigid the material, the more easily it will break. The structure of the material and its composition interfere directly in this parameter. A study [93] showed that chitosan nanocomposites reinforced with chitin whiskers increases the tensile strength

and significantly reduces the elongation at break. The work showed tensile strength of 52.23 MPa, and elongation at break of 21.32%.

Although microscopies of the films were not performed in this study, the incorporation of nanocellulose from CS, CH, and CC fibers into the films resulted in strong interactions between the plasticizer and the matrix modifying the mechanical profiles, which can demonstrate the compatibility between the phases. Similar results were identified by Marín–Silva et al. [42] when investigated chitosan nanocomposites with microcrystalline cellulose.

## **5. Conclusions**

The results found in the study confirm that the nanocellulose crystals of coconut shell (CS), corn husk (CH), and corncob (CC) fibers incorporated into the chitosan/glycerol films are presented as promising materials for the development of biodegradable composites. The crystallization by acid hydrolysis was favorable, resulting in nanocrystals with great potential to be used as reinforcement due to their size. Although the CS did not present the highest amount of extractable cellulose (12.50%), the highest concentration of nanocellulose in the dispersion after the acid hydrolysis was obtained from this fiber. In this study, wheat bran (WB) was not considered an interesting source of nanocrystals, which may be justified due to the low percentage of cellulose present in this residue.

Significant differences were observed in the properties of the film formulations studied, showing that the source of the nanocrystals is an important parameter to be investigated. The water activity varied from 0.601 (WB Film) to 0.658 (CH Film) and the moisture content from 15.13 (CS Film) to 20.86 (WB Film). The highest values for tensile strength were presented for CC (11.43 MPa) and CS (11.38 MPa) films. Therefore, the films prepared with the nanocrystals dispersion from the CS and CC presented an improvement of the mechanical properties, and the analysis of TEM indicated that both presented good characteristics to be applied in the development of nanobiocomposites.

**Author Contributions:** Conceptualization, J.I.D. and B.A.S.M.; Data curation, M.R.d.A., T.B.R.N., I.L.L., L.A.P.R., J.H.d.O.R. and B.A.S.M.; Formal analysis, M.R.d.A., T.I.d.S.e.S., I.L.L., L.A.P.R. and J.H.d.O.R.; Investigation, M.R.d.A., T.I.d.S.e.S., J.I.D. and B.A.S.M.; Methodology, M.R.d.A., T.B.R.N., T.I.d.S.e.S., I.L.L., J.H.d.O.R. and B.A.S.M.; Project administration, J.I.D. and B.A.S.M.; Resources, B.A.S.M.; Software, I.L.L. and L.A.P.R.; Supervision, J.I.D. and B.A.S.M.; Validation, T.B.R.N., L.A.P.R. and B.A.S.M.; Visualization, T.B.R.N. and J.H.d.O.R.; Writing—original draft, T.B.R.N., I.L.L., J.H.d.O.R., J.I.D. and B.A.S.M.

**Funding:** This research was funded by FAPESB (Foundation for Research Support of the State of Bahia) and SENAI CIMATEC, n◦ TSC018/2014.

**Acknowledgments:** The authors are grateful to FAPESB and SENAI CIMATEC for their financial support, to the Electron Microscopy Service of the Gonçalo Moniz Research Center - FIOCRUZ (Bahia) for the Transmission Electron Microscopy analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **E**ff**ect of Unbleached Rice Straw Cellulose Nanofibers on the Properties of Polysulfone Membranes**

**Mohammad Hassan 1,2,\*, Ragab E. Abou Zeid 1 , Wafaa S. Abou-Elseoud 1 , Enas Hassan 1 , Linn Berglund 3 and Kristiina Oksman 3,4,\***

Received: 5 May 2019; Accepted: 27 May 2019; Published: 29 May 2019

**Abstract:** In addition to their lower cost and more environmentally friendly nature, cellulose nanofibers isolated from unbleached pulps offer different surface properties and functionality than those isolated from bleached pulps. At the same time, nanofibers isolated from unbleached pulps keep interesting properties such as hydrophilicity and mechanical strength, close to those isolated from bleached pulps. In the current work, rice straw nanofibers (RSNF) isolated from unbleached neutral sulfite pulp (lignin content 14%) were used with polysulfone (PSF) polymer to make membrane via phase inversion. The effect of RSNF on microstructure, porosity, hydrophilicity, mechanical properties, water flux, and fouling of PSF membranes was studied. In addition, the prepared membranes were tested to remove lime nanoparticles, an example of medium-size nanoparticles. The results showed that using RSNF at loadings from 0.5 to 2 wt.% can significantly increase hydrophilicity, porosity, water flux, and antifouling properties of PSF. RSNF also brought about an increase in rejection of lime nanoparticles (up to 98% rejection) from their aqueous suspension, and at the same time, with increasing flux across the membranes. Tensile strength of the membranes improved by ~29% with addition of RSNF and the maximum improvement was obtained on using 0.5% of RSNF, while Young's modulus improved by ~40% at the same RSNF loading. As compared to previous published results on using cellulose nanofibers isolated from bleached pulps, the obtained results in the current work showed potential application of nanofibers isolated from unbleached pulps for improving important properties of PSF membranes, such as hydrophilicity, water flux, rejection, and antifouling properties.

**Keywords:** rice straw; cellulose nanofibers; unbleached pulp; polysulfone; membrane

## **1. Introduction**

Polysulfone (PSF) is one of the attractive polymers due to its good mechanical properties, thermal stability, chemical resistance, transparency, and flexibility [1]. PSF is used in different kinds of membranes such as anion-exchange membranes [2]; hemodialyzer membranes [3]; gas separation membranes [4]; pressure-driven membranes, i.e., ultra- and nanofiltration membranes [5]; osmotically-driven membranes for desalination and ions removal [6]; proton-exchange membranes fuel cells [7]; membranes for use in artificial organs [8]; and membranes used in dehydration of solvents by pervaporation [9].

To obtain membrane with sufficient porosity, the phase inversion technique, which depends on precipitation of PSF from its solution in a nonsolvent, is usually practiced. Nevertheless, the high hydrophobic nature of PSF leads to fouling problems when used in membranes [1]. To overcome this shortcoming, PSF-based polymers have been developed such as polyether sulfone and sulfonated PSF. This of course adds to the cost of the produced membranes. Some inorganic additives have been used to improve the hydrophilicity and performance of PSF membranes such as Al2O3 [10], TiO2 [11], and silica [12,13]. Another route to overcome the hydrophobicity of PSF and to produce high-flux membranes is to blend it with other relatively higher hydrophilic polymers. Compatibility between PSF and polymers used and their solubility are important factors which limit the number of candidates for that purpose. Different synthetic polymers such as polyvinylpyrrolidone [14], polyethylene glycol [15], and polyaniline-polyvinylpyrrolidone [16] have been used with PSF to improve the aforementioned shortcomings.

Cellulose, as a natural polymer, either in form of microfibers and nanocellulose, e.g., cellulose nanofibers and cellulose nanocrystals, have been studied to improve the hydrophilicity and water flux properties of PSF since they could be dispersed in the same solvents of PSF, such as dimethyl acetamide. The presence of S=O polar groups in polysulfones helps in forming hydrogen bonding with hydroxyl groups of cellulose. For example, cellulose nanocrystals have been used to improve hydrophilicity, mechanical properties, and performance of polysulfone [17–23] and polyether sulfone [24]. Regarding use of cellulose nanofibers (CNF), CNF isolated from bleached cellulose fibers have been used to improve mechanical and hydrophilicity of PSF [23], PSF/sulfonated PSF membrane [25], and polyether sulfone [26]. Also, methacryloxypropyltrimethoxy silane-modified CNF have been used to improve mechanical properties and performance of PSF membranes [27].

In addition to cellulose, the use of lignin, which produced as a byproduct from pulping processes, is another attractive route for improving hydrophilicity of polysulfones due to the presence of polar groups in the lignin structure in addition to the aromatic skeleton, which helps in achieving good compatibility between lignin and polysulfones [28,29].

Motivated by the desirable effect of both lignin and cellulose on properties of polysulfones, Ding et al. [30] studied the use of what they called "lignin/cellulose nanofibers" complex prepared by sulfuric acid hydrolysis of unbleached pulp followed by submerging the neutralized treated pulp in dimethylacetamide and high pressure homogenization, for improving hydrophilicity and mechanical properties of polyethersulfone; the ratio of lignin to cellulose nanofibers was up to 1.2 wt.%.

Furthermore, there is a recent interest in isolation of cellulose nanofibers from unbleached pulp with high lignin content using ultrafine grinding, where the nanofibers are isolated only by the action of the high shear force during grinding [31]. In addition to saving chemicals used in bleaching in case of using unbleached pulp, CNF with lignin at the surface could show good compatibility with polymers that do not have sufficient compatibility with nanofibers obtained from bleached pulp.

For the best of our knowledge, all previous work on using CNF with PSF for making membranes was mainly focused on using CNF isolated from bleached pulps. In the current work, the use of cellulose nanofibers isolated from unbleached rice straw neutral sulfite pulp (containing ~14% lignin) by ultrafine grinding for improving hydrophilicity, porosity, and ultrafiltration performance of PSF membrane was investigated. In addition, the effect of the isolated nanofibers on mechanical properties was investigated.

#### **2. Experimental**

#### *2.1. Materials*

Rice straw obtained from a local farm in Qalubiyah, Egypt was washed with water to remove the dust and allowed to air dry. Sodium sulfite and sodium carbonate used for pulping were reagent grade chemicals and used as received. Dimethylacetamide (DMAc) and polysulfone with average Mw ~35,000 and, average Mn ~16,000 were used as received from Sigmaaldrich (St. Louis, MO, USA). Lyophilised bovine serum albumin (pH 7) was purchased from Biowest Company (Biowest, Naillé, France) and used as received.

## *2.2. Preparation of Rice Straw Pulp*

Rice straw neutral sulfite pulp was prepared by pulping the straw using 10% sodium sulfite and 2% sodium carbonate (based on weight of rice straw) solutions at 160 ◦C for 2 h; the liquor ratio was 1:10. The produced pulp was thoroughly washed with water, defibrillated in a Valley beater (Valley Iron Works, Appleton, Wisconsin, USA) to a 25◦SR degree of freeness, dewatered, and allowed to air dry. Chemical composition of the prepared pulp: 16.63% ash content, 14.15% Klason lignin, 3.24% acid insoluble lignin, 54.12% α-cellulose, 14.34% pentosans, and degree of polymerization: 903 [31].

### *2.3. Xylanases Pretreatment of Unbleached Rice Straw Pulps*

Neutral sulfite unbleached pulp was pretreated with xylanases in citrate buffer (pH = 5.3) for 4 h at 50 ◦C as previously described [31]. The concentration of xylanase used was 0.04 g/g of pulp. Chemical composition of the pretreated pulp was 16.46% ash content, 13.18% Klason lignin, 2.31% acid insoluble lignin, 58.4% α-cellulose, 10.79% pentosans, and degree of polymerization: 1097 [31].

### *2.4. Isolation of Cellulose Nanofibers from Xylanase-Treated Unbleached Pulp*

Isolation of cellulose nanofibers from unbleached pulp was carried out similar to the previously published protocol [31]. In brief, the unbleached pulp was first disintegrated using a shear mixer (Silverson L4RT, Silverson Machines Ltd., Chesham, UK) using pulp suspension of 2 wt.% consistency. The pulp was then fibrillated using high-shear ultrafine friction grinder, or a so-called Supermasscolloider (MKCA6-2, Masuko Sangyo, Kawaguchi, Japan).The gap between the disks was gradually adjusted to −90 µm and the pulp was run through the grinder for approximately 140 min.

## *2.5. Preparation of PSF*/*RSNF Membrane*

PSF solution (18 wt.%) was prepared by dissolving in DMAc. Water in the RSNF suspension was first removed by vacuum filtration then acetone was passed once through the filtered CNF, and finally DMAc was passed twice. The DMAc-wetted RSNF were kept in a closed container in fridge at 8 ◦C till use. The DMAc-wetted RSNF were added to PSF solution at ratios from 0.5 to 2 wt.% of dry RSNF to PSF; the mixture was homogenized by magnetic stirring for 30 min. The viscosity of the PSF/RSNF mixture was measured using a tuning-fork vibration viscometer (Vibro Viscometer SV-10, A&D Company Limited, Tokyo, Japan). The films were prepared by phase inversion in distilled water, washed thoroughly with distilled water, and left to dry in air.

## *2.6. Characterization of PSF*/*RSNF Membrane*

Tensile testing was carried out on 1-cm-wide films using a Lloyd instrument (Lloyd Instruments, West Sussex, UK) with a 100-N load cell; a cross-head speed of 2 mm/min was used and the gauge length was 20 mm. Five replicates of each sample were used and the results averaged. The water contact angle of films was measured using an Attension theta lite measuring system (Biolin Scientific AB, Gothenburg, Sweden) and calculated with the drop shape analysis OneAttension Version 2.7 (r5433), using a sessile drop technique. A 4 µL water drop was placed onto the films at four separate places for calculating the average contact angles. Microscopic features of films were investigated using a FEI Quanta 200 scanning electron microscope (FEI Company, Eindhoven, The Netherlands) at an acceleration voltage of 20 kV.

### *2.7. Preparation and Characterization of Lime Nanoparticles Suspension*

Lime nanoparticles were prepared according to the previously published method [32]. In brief, to 100 mL containing 0.3 mol/L of calcium chloride, 0.6 mol/L of sodium hydroxide was added

dropwise (≈4 mL/min) at 90◦C. The precipitated calcium hydroxide nanoparticles were centrifuged and washed with previously boiled distilled water. The suspension containing purified calcium hydroxide nanoparticles was flushed with nitrogen gas and kept in closed bottle until use. Transmission electron microscopy (TEM) was carried out using high-resolution transmission electron microscopy (HR-TEM) (JEM-2100 transmission electron microscope, JEOL, Tokyo, Japan). Energy-dispersive X-ray microanalysis was carried out using JEOL JXA 8040 A electron probe microanalyzer (JEOL, Tokyo, Japan).

#### *2.8. Evaluation of Membranes Properties*

#### 2.8.1. Porosity

The porosity (ε) of membranes was determined from water absorption of the different membranes. The membranes were submerged in distilled water for 18 h then weighed after wiping excess water. Then, the wet membranes were dried at 105 ◦C until no change in weight. Porosity was calculated according to the following equation [33].

$$\text{Porosity } (\varepsilon) = [(\mathbf{m}\_1 - \mathbf{m}\_2) \langle \rho.\text{A.L} \rangle^\* 100\tag{1}$$

where m1 and m2 are the weight of the wet and dry films, respectively; % is the water density (g/cm3 ); A is the effective area of the films (cm2 ); and L is the film thickness (cm).

#### 2.8.2. Pure Water Flux and Fouling

The water flux of the membranes was measured using a dead-end stirred cell (Sterlitech HP4750, Sterlitech, Kent, WA, USA). Prior to the measurements, discs with a diameter of ~5 cm were cut out from the membranes and soaked in water for one hour to ensure equilibration of the membrane. The conditioned membranes were placed in the dead-end cell on a stainless steel porous support disc and water was passed through the membranes at 25 ◦C at a differential pressure of 30 Mpa, maintained using pressure water pump. The quantity of water that passed through the membrane for a defined time interval was weighed accurately and the flux was calculated (L/h/m2 /MPa) for the active filtration area (14.6 cm2 ). To avoid reduction of water flux as a result of membrane compaction, back pressure was applied every 10 min.

To test fouling of the membranes, bovine serum albumin solution (1 g/L) was passed through the membrane for 10 min under pressure of 30 MPa and flux was calculated. Then, the membranes were washed briefly with distilled water and pure water was passed through the membranes under the same pressure and protein solution was passed again; the cycle was repeated for one hour.

#### 2.8.3. Removing Lime Nanoparticles

To test the efficiency of the prepared membranes in removing lime, suspensions containing 1 g/L was passed through the membrane under the same conditions mentioned above for testing pure water flux. The concentration of the nanoparticles in the filtrate was measured from following the turbidity using UV–Visible spectrometer (Shimadzu, Tokyo, Japan) at wavelength of 600 nm. Turbidity (T) was calculated from measuring absorbance at 500 nm: Turbidity = (A \* 2.302)/L, where A is the absorbance and l is the path length (0.01 m). The rejection of the prepared membranes to remove the nanoparticles was calculated using the following formula.

$$\text{Rejection } (\%) = [(\text{Control } \text{T}\_{500} - \text{sample } \text{T}\_{500})(\text{Control } \text{T}\_{500}) \times 100 \tag{2}$$

#### **3. Results and Discussion**

In a previous publication, it has been shown that RSNF with high lignin content (~14 wt.%) could be isolated from rice straw xylanase-treated unbleached sulfite pulp with a width of ~14 ± 7 nm, as seen in Figure 1 [31]. Water in the isolated RSNF could be easily exchanged by filtration and simple washing using di *Polymers* **2019** fferent non-aqueous aprotic solvent [34]. , *11*, 938 5 of 14

**Figure 1.** TEM image of cellulose nanofibers isolated from xylanase-treated rice straw unbleached neutral sulfite pulp. **Figure 1.** TEM image of cellulose nanofibers isolated from xylanase-treated rice straw unbleached neutral sulfite pulp.

#### *3.1. Effect of RSNF on Viscosity of PSF Solution 3.1. E*ff*ect of RSNF on Viscosity of PSF Solution*

The effect of the addition of RSNF on viscosity of the PSF solution was studied as an indication of good dispersion of RSNF in the solution; the results are presented in Table 1. As shown in the figure, the viscosity of PSF solution increased with the addition of RSNF, in spite of the small concentrations of RSNF added (2% max.). The increase in viscosity ranged from 23% to 128% at the different RSNF loadings with respect to neat PSF solution. The effect of the addition of RSNF on viscosity of the PSF solution was studied as an indication of good dispersion of RSNF in the solution; the results are presented in Table 1. As shown in the figure, the viscosity of PSF solution increased with the addition of RSNF, in spite of the small concentrations of RSNF added (2% max.). The increase in viscosity ranged from 23% to 128% at the different RSNF loadings with respect to neat PSF solution.

**Table 1.** Effect of rice straw nanofibers (RSNF) content on viscosity of polysulfone (PSF) solution. **Table 1.** Effect of rice straw nanofibers (RSNF) content on viscosity of polysulfone (PSF) solution.

#### Microscopic structure of polymeric membranes is very important since it determines both *3.2. E*ff*ect of RSNF on Microscopic Structure of PSF Membranes*

mechanical and filtration properties. The effect of RSNF on the microscopic structure of PSF membranes formed by phase inversion was studied by SEM; images of both surface and cross-section are shown in Figures 2 and 3. Regarding the cross-section, a slight change in the size of voids started to occur at RSNF loading of 1%, while at 2%, formation of larger and elongated slender-like voids was observed. On the other Microscopic structure of polymeric membranes is very important since it determines both mechanical and filtration properties. The effect of RSNF on the microscopic structure of PSF membranes formed by phase inversion was studied by SEM; images of both surface and cross-section are shown in Figures 2 and 3.

hand, the microscopic structure of PSF film' surfaces clearly affected by the addition of RSNF. While neat PSF film had a few and very tiny pores at the surface, adding RSNF resulted in more and wider pores, especially at RSNF loading 1–2%. At higher magnification, SEM images of the surface of membrane sample containing 2% RSNF showed that although wide pores formed at the surface, the inner pores were narrower and with mesh-like structure. Table 2 shows the average diameter of the pores at the surface of the different PSF samples seen from the images. Increasing the loading of RSNF resulted in larger pore diameter at the surface. Regarding the cross-section, a slight change in the size of voids started to occur at RSNF loading of 1%, while at 2%, formation of larger and elongated slender-like voids was observed. On the other hand, the microscopic structure of PSF film' surfaces clearly affected by the addition of RSNF. While neat PSF film had a few and very tiny pores at the surface, adding RSNF resulted in more and wider pores, especially at RSNF loading 1–2%. At higher magnification, SEM images of the surface of membrane sample containing 2% RSNF showed that although wide pores formed at the surface, the inner pores were narrower and with mesh-like structure.

Similar trend was observed in membranes prepared from cellulose nanofibers isolated from bleached pulp with polysulfone/sulfonated polysulfone mixture [24]. These changes in the microscopic features due to addition of cellulose nanofibers were interpreted by the accelerated phase inversion process by the hydrophilic cellulose nanofibers, which results in formation of more pores and better pores connectivity across the membrane. In addition, due to the large aspect ratio of cellulose Table 2 shows the average diameter of the pores at the surface of the different PSF samples seen from the images. Increasing the loading of RSNF resulted in larger pore diameter at the surface. Similar trend was observed in membranes prepared from cellulose nanofibers isolated from bleached pulp with polysulfone/sulfonated polysulfone mixture [24]. These changes in the microscopic features due

nanofibers, they are easily aggregated during phase inversion and resulted in formation of pore

to addition of cellulose nanofibers were interpreted by the accelerated phase inversion process by the hydrophilic cellulose nanofibers, which results in formation of more pores and better pores connectivity across the membrane. In addition, due to the large aspect ratio of cellulose nanofibers, they are easily aggregated during phase inversion and resulted in formation of pore defects in the membranes [18,25]. The same findings were also reported when methacryloxypropyltrimethoxy silane-modified cellulose nanofibers were used with PSF [27]. **Table 2.** Diameter of pores at the surface of PSF/RSNF films. **Sample Diameter of pores at surface (µm)**  PSF 2.9 ± 1.2 PSF + 0.5% RSNF 5.9 ± 3.4 PSF + 1.0% RSNF 5.8 ± 0.8 PSF + 2.0% RSNF 15.3 ± 8.6

*Polymers* **2019**, *11*, 938 6 of 14

methacryloxypropyltrimethoxy silane-modified cellulose nanofibers were used with PSF [27].

**Figure 2. Figure 2.** SEM of cross-section of polysulfone membranes with different RSNF loadings. SEM of cross-section of polysulfone membranes with different RSNF loadings.

*Polymers* **2019**, *11*, 938 7 of 14

**Figure 3.** SEM image of surfaces of PSF (**a**–**c**), PSF/0.5% RSNF (**d**–**f**), PSF/1% RSNF (**g**–**i**), and PSF/2% RSNF (**j**–**l**) membranes at 800, 3000, and 12000x magnification, respectively (scale bar is 50, 40, and 10 µm, respectively). **Figure 3.** SEM image of surfaces of PSF (**a**–**c**), PSF/0.5% RSNF (**d**–**f**), PSF/1% RSNF (**g**–**i**), and PSF/2% RSNF (**j**–**l**) membranes at 800, 3000, and 12000x magnification, respectively (scale bar is 50, 40, and 10 µm, respectively).

#### *3.3. Effect of RSNF on Mechanical Properties of PSF Films 3.3. E*ff*ect of RSNF on Mechanical Properties of PSF Films*

The effect of RSNF on tensile strength properties of PSF was studied and results are listed in Table 3. As the results show, adding RSNF resulted in a moderate increase in maximum tensile strength (~29%) at 0.5% RSNF loading followed by a decrease at higher RSNF loading, but still as high as that of neat PSF. This could be attributed to the more pores formed at the surface and pore defects across the thickness. The high standard deviations values at 2% RSNF loading can be attributed to the high porosity of the films, and thus failure of films was non-reproducible from one sample to another. On the other hand, the Young's modulus of PSF films more obviously increased upon adding RSNF, especially at 0.5% loading of RSNF where the increase in modulus was ~40%. Increasing RSNF to higher loadings resulted in decreasing the modulus values, but they were still higher than that of the neat PSF membrane. The improvement in mechanical properties of the films indicates good compatibility between PSF matrix and the surface of RSNF which are rich in lignin with its aromatic ring structures. In addition, formation of hydrogen bonding between hydroxyl The effect of RSNF on tensile strength properties of PSF was studied and results are listed in Table 3. As the results show, adding RSNF resulted in a moderate increase in maximum tensile strength (~29%) at 0.5% RSNF loading followed by a decrease at higher RSNF loading, but still as high as that of neat PSF. This could be attributed to the more pores formed at the surface and pore defects across the thickness. The high standard deviations values at 2% RSNF loading can be attributed to the high porosity of the films, and thus failure of films was non-reproducible from one sample to another. On the other hand, the Young's modulus of PSF films more obviously increased upon adding RSNF, especially at 0.5% loading of RSNF where the increase in modulus was ~40%. Increasing RSNF to higher loadings resulted in decreasing the modulus values, but they were still higher than that of the neat PSF membrane. The improvement in mechanical properties of the films indicates good compatibility between PSF matrix and the surface of RSNF which are rich in lignin with its aromatic ring structures. In addition, formation of hydrogen bonding between hydroxyl groups at the surface of the RSNF and the S=O bonds of PSF could improve mechanical properties of the films. Regarding strain at break, it generally tended to decrease with addition of RSNF; the decrease ranged from 14.5 to 20.7%.

Comparing the improvement in tensile strength properties in the current work to that found in previous work where the so-called "lignin/cellulose nanofibers" complex [30], prepared by sulfuric acid hydrolysis of unbleached pulp and high pressure homogenization in dimethylacetamide, was used with PSF, the maximum improvement in tensile strength was about 50%. Strain at break increased by ~22%, which is in contrast to the strain decrease found in the current work. The tensile modulus was not measured in that work. In another work where cellulose nanofibers from bleached pulp were used with PSF and sulfonated PSF, the maximum improvement in tensile strength and tensile modulus were approximately 20% and 38%, respectively [25]. Comparing the improvement in tensile strength and tensile modulus achieved in the current work to the aforementioned findings clearly shows the advantage of using RSNF isolated from unbleached pulp by direct grinding.

**Table 3.** Tensile strength properties of polysulfone (PSF)/rice straw nanofibers (RSNF) membranes.

## *3.4. E*ff*ect of RSNF on Hydrophilicity and Porosity of PSF Membranes*

One of the important reasons for using cellulose in membranes is to increase their hydrophilicity; this affects both water flux across the membrane and its resistance to fouling. The RSNF used still has strong hydrophilicity in spite of the presence of lignin. As shown in Table 4, addition of RSNF to PSF resulted in a decrease in water contact angle even at the smallest RSNF loading where water contact angle was ~90◦ for the neat PSF membrane and ranged from 80.1◦ to 82.7◦ for the different PSF/RSNF samples. The effect of RSNF on water absorption, and thus the calculated porosity, of PSF film was rather more pronounced at 1–2% of RSNF loading. PSF film has poor hydrophilic character and water absorption occurred is mainly due to permeation of water into the porous structure. Increasing porosity as a result of adding RSNF could be due to the higher hydrophilicity, as it is clear from the contact angle values, and wider pores formed at the surface of PSF as a result of presence of RSNF as seen from the SEM images.

**Table 4.** Water contact angle of the different PSF/RSNF membranes.

## *3.5. E*ff*ect of RSNF on Water Flux and Fouling of PSF Films*

Water flux across PSF and PSF/RSNF membranes was tested and the results are presented in Figure 4. Cycles of back pressure were applied every twenty minutes to minimize the decrease of flux as a result of compactness of the film by water pressure. As it is clear in the figure, presence of RSNF with loading 1% and higher had significant effect on water flux. After one hour of the experiment,

water flux values of 50 ± 5, 57 ± 5, 111 ± 18, and 131 ± 21 L/h/m2 /MPa were recorded for neat PSF, PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2%RSNF, respectively. The increase in water flux could be attributed to the higher hydrophilicity, porosity, and water absorption of membrane with higher RSNF contents. The increase in water flux from cycle to another could be due to generation of more pores at the surface by the action of water pressure applied. This is could be seen from the SEM images of membranes' surface before and after the water flux test (Figure 5). As indicated by the red arrows in the image, the skin layer at the surface was ruptured by the action of water pressure applied generating more pores at the surface. water flux values of 50 ± 5, 57 ± 5, 111 ± 18, and 131 ± 21 L/h/m2/MPa were recorded for neat PSF, PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2%RSNF, respectively. The increase in water flux could be attributed to the higher hydrophilicity, porosity, and water absorption of membrane with higher RSNF contents. The increase in water flux from cycle to another could be due to generation of more pores at the surface by the action of water pressure applied. This is could be seen from the SEM images of membranes' surface before and after the water flux test (Figure 5). As indicated by the red arrows in the image, the skin layer at the surface was ruptured by the action of water pressure applied generating more pores at the surface. water flux values of 50 ± 5, 57 ± 5, 111 ± 18, and 131 ± 21 L/h/m2/MPa were recorded for neat PSF, PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2%RSNF, respectively. The increase in water flux could be attributed to the higher hydrophilicity, porosity, and water absorption of membrane with higher RSNF contents. The increase in water flux from cycle to another could be due to generation of more pores at the surface by the action of water pressure applied. This is could be seen from the SEM images of membranes' surface before and after the water flux test (Figure 5). As indicated by the red arrows in the image, the skin layer at the surface was ruptured by the action of water pressure applied generating more pores at the surface.

*Polymers* **2019**, *11*, 938 9 of 14

*Polymers* **2019**, *11*, 938 9 of 14

**Figure 4.** Water flux of PSF and PSF/RSNF membranes with different loadings of RSNF. **Figure 4.** Water flux of PSF and PSF/RSNF membranes with different loadings of RSNF. **Figure 4.** Water flux of PSF and PSF/RSNF membranes with different loadings of RSNF.

**Figure 5.** SEM of the PSF/2%RSNF membrane surface: (**a**) after water flux test and (**b**) before water flux test. **Figure 5.** SEM of the PSF/2%RSNF membrane surface: (**a**) after water flux test and (**b**) before water flux test. **Figure 5.** SEM of the PSF/2%RSNF membrane surface: (**a**) after water flux test and (**b**) before water flux test.

Regarding fouling of the membranes, as shown in Figure 6, after three cycles of passing protein solution and pure water across the membranes for one hour, water flux values of 2.7 ± 0.26, 8.9 ± 1.75, 18.6 ± 2.48, and 61.3 ± 4.4 L/h/m2/MPa were recorded for neat PSF, PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2%RSNF, respectively. Regarding fouling of the membranes, as shown in Figure 6, after three cycles of passing protein solution and pure water across the membranes for one hour, water flux values of 2.7 ± 0.26, 8.9 ± 1.75, 18.6 ± 2.48, and 61.3 ± 4.4 L/h/m2/MPa were recorded for neat PSF, PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2%RSNF, respectively. Regarding fouling of the membranes, as shown in Figure 6, after three cycles of passing protein solution and pure water across the membranes for one hour, water flux values of 2.7 ± 0.26, 8.9 ± 1.75, 18.6 ± 2.48, and 61.3 ± 4.4 L/h/m2 /MPa were recorded for neat PSF, PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2%RSNF, respectively.

*Polymers* **2019**, *11*, 938 10 of 14

**Figure 6.** Fouling of PSF and PSF/RSNF membranes with different loadings of RSNF when passing 1% bovine albumin solution. **Figure 6.** Fouling of PSF and PSF/RSNF membranes with different loadings of RSNF when passing 1% bovine albumin solution. **Figure 6.** Fouling of PSF and PSF/RSNF membranes with different loadings of RSNF when passing Time (h)

#### *3.6. Rejection of Lime Nanoparticles 3.6. Rejection of Lime Nanoparticles* 1% bovine albumin solution.

The prepared membranes were tested for removing lime nanoparticles from dilute suspension containing 2 g/l of lime nanoparticles. The XRD pattern (Figure 7) showed that structure of calcium hydroxide (lime) with hexagonal crystal structure [35]; TEM image showed nanoparticles with diagonal ranged from as low as 100 nm up to several hundred nanometers. The energy-dispersive Xray spectroscopy (EDS) spectrum proved the purity of the prepared lime nanoparticles. The prepared membranes were tested for removing lime nanoparticles from dilute suspension containing 2 g/L of lime nanoparticles. The XRD pattern (Figure 7) showed that structure of calcium hydroxide (lime) with hexagonal crystal structure [35]; TEM image showed nanoparticles with diagonal ranged from as low as 100 nm up to several hundred nanometers. The energy-dispersive X-ray spectroscopy (EDS) spectrum proved the purity of the prepared lime nanoparticles. *3.6. Rejection of Lime Nanoparticles*  The prepared membranes were tested for removing lime nanoparticles from dilute suspension containing 2 g/l of lime nanoparticles. The XRD pattern (Figure 7) showed that structure of calcium hydroxide (lime) with hexagonal crystal structure [35]; TEM image showed nanoparticles with diagonal ranged from as low as 100 nm up to several hundred nanometers. The energy-dispersive X-

ray spectroscopy (EDS) spectrum proved the purity of the prepared lime nanoparticles.

1000

**Figure 7.** TEM, XRD pattern, and energy-dispersive X-ray spectroscopy (EDS) spectrum of lime nanoparticles.

The water flux of the lime nanoparticle suspension after 2 h for the different membranes is shown in Table 5. As shown in the table, adding the RSNF resulted in increasing the flux of lime nanoparticles suspension. The maximum increase of water flux was achieved on adding 1–2% of RSNF and was ~850% as compared to that of the neat PSA membrane.

Figure 8 shows visible light absorbance of the filtrate solution after passing through PSF membranes, as well as that of lime nanoparticles suspension with 1 g/L concentration. Interestingly, as shown in the figure, the rejection of lime nanoparticles increased with increasing RSNF content in spite of the increase of diameter of the pores at the surface and porosity of the membranes containing RSNF. The filtrate obtained from filtration using the neat PSF membrane showed a 60% decrease of light absorbance at 600 nm as compared to the blank lime suspension; filtrates obtained from filtration using PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2% RSNF showed 76%, 97%, and 98% decrease in light absorbance, respectively. According to standard curve of lime nanoparticles with different concentrations (not shown), the concentration of lime nanoparticles in the filtrate was 17.4, 11.1, 1.3, and 0.9 mg/L when using PSF, PSF/0.5%RSNF, PSF/1%RSNF, and PSF/2% RSNF membranes, respectively. The increase in rejection of lime nanoparticles with increasing porosity and pore radius at the surface of membranes as the RSNF content increase means that the rejection of lime nanoparticles is not simply by mechanical filtration but involves interaction between the nanoparticles and the functional groups at the RSNF surfaces. RNSF with its very high surface area has plenty of hydroxyl and carboxylic functional groups at their surface which can attract the charged lime nanoparticles. Similar observation was found in previous work in case of using microcrystalline cellulose, cellulose nanocrystals or cellulose nanofibers isolated from bleached pulps when rejection of bovine serum albumin (BSA) was studied [25,27,36]. Although addition of cellulose nanocrystals or nanofibers to PSF resulted in increasing porosity and water flux, rejection of BSA (protein with charged surface) did not significantly affect by the increased porosity or water flux. *Polymers* **2019**, *11*, 938 12 of 14

**Figure 8.** Absorbance spectra of lime nanoparticles suspension and filtrate produced after passing the **Figure 8.** Absorbance spectra of lime nanoparticles suspension and filtrate produced after passing the suspension through the different PSF membranes.

suspension through the different PSF membranes.

**4. Conclusions**

M.H. and K.O.

**References**

appointed by the Swedish government.

https://doi.org/10.1002/0471440264.

*Sci. Technol.* **2017**, *8*, 183–196.

**Conflicts of Interest:** The authors declare no conflicts of interest.

showed the advantages of using cellulose nanofibers isolated with lower cost than that isolated from bleached pulps for improving properties of PSF related to their use as ultrafiltration membranes.

**Author Contributions:** Conceptualization, M.H. and K.O.; Funding Acquisition, M.H. and K.O.; Investigation, M.H., R.E.A.Z., W.S.A.-E., E.H., L.B., and K.O.; Methodology, M.H., R.E.A.Z., W.S.A.-E., E.H., and L.B.; Project Administration, K.O.; Supervision, M.H. and K.O.; Writing—Original Draft, M.H.; Writing—Review & Editing,

**Funding:** The authors acknowledge funding of the current research by the Swedish Research Council (project no. 2015-05847), as well as the financial support received from Bio4Energy, a strategic research environment

1. Mark, H.F. Encyclopedia of Polymer Science and Technology. *John Wiley & Sons*, 2002.

2. Cho, M.K.; Lim, A.; Lee, S.Y.; Kim, H.-J.; Yoo, S.J.; Sung, Y.-E.; Park, H.S.; Jang, J.H. A Review on Membranes and Catalysts for Anion Exchange Membrane Water Electrolysis Single Cells. *J. Electrochem.*

RSNF containing lignin (isolated from unbleached pulp) could be used for improving porosity, hydrophilicity, water flux, and antifouling of PSF membranes at low loadings of RSNF (0.5–2%). Mechanical properties (tensile stress and Young' modulus) only improved at the lowest RSNF loading (0.5%); higher loadings did not enhance these properties. PSF containing 1–2% of RSNF could

**Table 5.** Flux of lime nanoparticles suspension through the different membranes after 2 h.

#### **4. Conclusions**

RSNF containing lignin (isolated from unbleached pulp) could be used for improving porosity, hydrophilicity, water flux, and antifouling of PSF membranes at low loadings of RSNF (0.5–2%). Mechanical properties (tensile stress and Young' modulus) only improved at the lowest RSNF loading (0.5%); higher loadings did not enhance these properties. PSF containing 1–2% of RSNF could be successfully used for removing lime nanoparticles from aqueous suspension at a 10-fold faster rate than in case of using neat PSF, which could only partially remove the lime nanoparticles. The study showed the advantages of using cellulose nanofibers isolated with lower cost than that isolated from bleached pulps for improving properties of PSF related to their use as ultrafiltration membranes.

**Author Contributions:** Conceptualization, M.H. and K.O.; Funding Acquisition, M.H. and K.O.; Investigation, M.H., R.E.A.Z., W.S.A.-E., E.H., L.B., and K.O.; Methodology, M.H., R.E.A.Z., W.S.A.-E., E.H., and L.B.; Project Administration, K.O.; Supervision, M.H. and K.O.; Writing—Original Draft, M.H.; Writing—Review & Editing, M.H. and K.O.

**Funding:** The authors acknowledge funding of the current research by the Swedish Research Council (project no. 2015-05847), as well as the financial support received from Bio4Energy, a strategic research environment appointed by the Swedish government.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Poly(3-hydroxybutyrate) Modified by Plasma and TEMPO-Oxidized Celluloses**

**Denis Mihaela Panaitescu 1,\* , Sorin Vizireanu 2,\* , Sergiu Alexandru Stoian 1,3 , Cristian-Andi Nicolae 1 , Augusta Raluca Gabor 1 , Celina Maria Damian 3 , Roxana Trusca 4 , Lavinia Gabriela Carpen 2,5 and Gheorghe Dinescu 2,5**

Received: 22 June 2020; Accepted: 6 July 2020; Published: 7 July 2020

**Abstract:** Microcrystalline cellulose (MCC) was surface modified by two approaches, namely a plasma treatment in liquid using a Y-shaped tube for oxygen flow (MCC-P) and a TEMPO mediated oxidation (MCC-T). Both treatments led to the surface functionalization of cellulose as illustrated by FTIR and XPS results. However, TEMPO oxidation had a much stronger oxidizing effect, leading to a decrease of the thermal stability of MCC by 80 ◦C. Plasma and TEMPO modified celluloses were incorporated in a poly(3-hydroxybutyrate) (PHB) matrix and they influenced the morphology, thermal, and mechanical properties of the composites (PHB-MCC-P and PHB-MCC-T) differently. However, both treatments were efficient in improving the fiber–polymer interface and the mechanical properties, with an increase of the storage modulus of composites by 184% for PHB-MCC-P and 167% for PHB-MCC-T at room temperature. The highest increase of the mechanical properties was observed in the composite containing plasma modified cellulose although TEMPO oxidation induced a much stronger surface modification of cellulose. This was due to the adverse effect of more advanced degradation in this last case. The results showed that Y-shaped plasma jet oxidation of cellulose water suspensions is a simple and cheap treatment and a promising method of cellulose functionalization for PHB and other biopolymer reinforcements.

**Keywords:** cellulose; plasma in liquids; polyhydroxyalkanoate; polymer composites; thermal properties; DMA

## **1. Introduction**

Biopolymers are seen as a viable alternative to synthetic polymers in many applications and they are unparalleled in the medical field [1–3]. Poly(3-hydroxybutyrate) (PHB) is an aliphatic polyester obtained by microbial synthesis and, for the moment, is the most affordable of polyhydroxyalkanoates (PHAs) family. The known shortcomings of PHB are the high cost and brittleness along with the low thermal stability during melt processing [4,5]. Adding plasticizers, elastomers, and organic or inorganic fillers are the most used strategies to improve PHB properties [6–8]. Among the fillers, microand nano-celluloses were most intensely studied to modify PHB and other aliphatic polyesters [9–12].

Microfibrillated cellulose is obtained from cellulose, a sustainable and low-cost naturally occurring biopolymer. This is the most abundant organic polymer on Earth and comprises 33% of all the plants on the planet [13,14]. Microfibrillated cellulose is biodegradable and biocompatible and has reduced carbon dioxide emissions in the environment. It shows an adaptable surface chemistry and a unique spectrum of properties: high water-uptake capability, low density, high crystallinity and Young's modulus, and good thermal stability [12–14]. The research, production, and application of microfibrillated cellulose have experienced a real boost in the last twenty years. It can be extracted from a multitude of sources, such as wood, plants, algae, by-products, and waste of the agro-food industry by mechanical disintegration [13–15]. Refining, homogenization, high power ultrasonication, or microfluidization, with or without additional enzymatic or chemical pretreatments are generally the most used methods to obtain long flexible microfibrils [12–15].

TEMPO-mediated oxidation of cellulose consists of surface oxidation of cellulose in aqueous suspension using sodium hypochlorite as oxidant and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) and sodium bromide as catalysts at an alkaline pH [16–18]. This pre-treatment proved to be a successful method for ensuring the selective conversion of C6 hydroxyl group of cellulose to carboxylate group. Previous studies have shown the great advantage of this pre-treatment which reduces the energy consumption and the number of cycles in the subsequent stages of mechanical defibrillation [18,19].