Rubber particle size in RRIM600 and RRIT251 clones are found to be larger in the range between 0.50 μm and 4.50 μm. The fine particles with size lower than 0.50 μm are observed in BPM24 clone together with more portions of larger rubber particles in the range between 0.40 μm and 3.50 μm and shows wider particle size distribution than other clones, whereas PB235 clone shows narrower distribution in the range of 0.45–2.52 μm. The difference in the range of particle size might be attributed to the non-rubbers and the unique signature of the clones [20]. These results are in good agreement with the results of transmission electron micrographs as shown in Figure 3. It is clearly observed that the latex of RRIM600 and RRIT251 clones consist of large particles (Figure 3a,b), while the latex from PB235 and BPM24 clones (Figure 3c,d) consist of both large and small particles. Rubber particles are observed as spherically shaped and some were pear-shaped; regardless, their particle size, either they were large or small rubber particles for all clones selected [21]. It was observed that some elongated particles appeared as a result of aggregation of non-rubber in the analyzed sections of BPM24, consisting of many nodules of non-rubber on the particle surfaces.

**Figure 3.** Transmission electron micrographs of (**a**) RRIM600; (**b**) RRIT251; (**c**) PB235; and (**d**) BPM24. (×10,000).

#### *3.3. Molar Mass Distribution of the Fresh Natural Latex*

The molar mass and molar mass distributions (MMD) in the natural rubber play an important role in their bulk properties. The molar mass distribution of natural rubber from RRIM600, RRIT251, PB235 and BPM24 clones are shown in Table 4. MMD, in the case of all clones, confirmed that the rubber from RRIM600 and RRIT251 are bimodal MMD, but the rubber from PB235 and BPM24 clones are unimodal MMD. The properties of NRs from the four clones depend on the relative quantity of short chains. It is anticipated that the high number of short chains in this bimodal distribution yielded many chain ends or terminals of NR molecules [10]. The polydispersity index (PDI) of the BPM24 clone exhibited a superior value when compared to the other clones and exhibited an MWD curve with unimodal distribution with almost constant value skewed in case of low molecular weight. However, the size of rubber particles might influence the molecular weight of their rubber particles in the latex.

**Table 4.** Molecular weight and polydispersity index of fresh natural latex from four different *Hevea brasiliensis* clones.

#### *3.4. Mechanical Properties of Fresh Natural Latex*

Table 5 shows the mechanical properties of NR films collected from four different *Hevea brasiliensis* clones. The green strength of elastomers has been commonly attributed to long-chain branching, interactions between polar groups, the presence of a gel, chain entanglements, and crystallization on stretching [22]. The variations in these factors are responsible for the different green strength in the case of NRs from various rubber clones. It was seen that RRIM600, RRIT251 and BPM24 clones showed higher stress at break compared to PB235. This might be attributed to the lower protein content (Table 3) that corresponds to the levels of short chains *cis-1,4*-isoprene. It was presumed that the non-rubber components in rubber molecules caused the formation of loosely crosslinked structures or a gel in natural rubber. Generally, the area under stress-strain curve indicates the toughness of a material. In Figure 4, it is seen that the PB235 clone showed the least area due to the least toughness among the other clones, and this was related to the results reported previously [10,11]. This finding clearly indicates that the non-rubber components (i.e., protein content) acted as the reinforcing filler and enhanced the rigidity of rubber. They also form macro-gel in rubber molecules by providing stronger rubber networks with high moduli, stiffness, and hardness [11,23–25]. This indicates that the removal of proteins or non-rubber components lead to reduce the moduli, stress at break and hence the stiffness of rubber [25]. In the previous work, [10,22,26] rubber molecular chains comprise of long-chain isoprene units and the chain ends consist of one protein end group and another phospholipid end group. It can be seen that the α-terminal group with mono- or di-phosphate groups associated with phospholipids, whereas the ω-terminal is a dimethylallyl group that interacts with proteins [26]. In Figure 5, it is clearly seen that these molecules could interact with both the functional terminal groups via hydrogen bonding and ionic bonds derived from metal ions. The proteins in natural rubber are considered to originate branch points by hydrogen bonding, as well as the phospholipids are linked to another phospholipid molecule in other chain ends via hydrogen bonding or ionic bonds derived from metal ions. Moreover, proteins at ω-terminal could interact with other protein molecules through hydrogen bonding [10,26]. In addition, the increase in green strength can be attributed to the number of branch points per chain and chain entanglement. The long chain branching in rubber molecule plays an important role in the higher green strength [22]. It was found

that a high polydispersity index in case of RRIM600 and BPM24 clones exhibited the higher green strength. These findings might have resulted from the distribution of larger rubber particles and not that of smaller particles. Thus, it is reasonable to assume that the long chain branching in natural rubber affects the green strength. Therefore, it is concluded that the green strength is related to the non-rubber components, branch structure, chain entanglements, and particle size in the natural rubber molecules.

**Table 5.** Mechanical properties of NR from fresh natural latex collected from four different *Hevea brasiliensis* clones.

**Figure 4.** Stress-strain curves of NRs from the four *Hevea brasiliensis* clones.

**Figure 5.** Schematic representation of α- and ω-terminal groups on the rubber latex particle surface.

#### *3.5. Characterization of Cream Concentrated Latex from Four Different Hevea Brasiliensis Clones*

Creaming method is a physicochemical process in which the creaming agent is mixed with fresh natural latex and then a phase separation process (upper rubber fraction and lower serum) is applied [4]. This work is aimed at preparing concentrated latex using hydroxyethyl cellulose (HEC) as a creaming agent. Creamed concentrated latex generally contains the creaming agent, as well as ammonia for preservation. It was found that TSC, DRC, viscosity, and particle size values of the cream concentrated latex are clearly higher than those of fresh natural latex, whereas the protein content, surface tension, and the color exhibited lower values. Table 6 shows the TSC and DRC of creamed concentrated latex, which are increased in the case of all clones when compared to the fresh natural latex. This indicates that hydroxyethyl cellulose (HEC) increases the efficiency to separate rubber particles from the serum solution [2,4]. The incorporation of high-molecular weight hydroxyethyl cellulose (HEC) with higher degree of polarity is responsible for the creaming phenomenon. Thus, hydroxyethyl cellulose (HEC) as a hydrophilic colloid dispersed in aqueous medium and covered the surface of rubber particles. Also, the branched segments of the HEC molecules could be well entangled with the neighboring rubber particles (Figure 6), restricting the movement of the rubber particles and causing a larger rubber particle size that led to an increase in viscosity of the latex [2,27]. It is clear that the creamed concentrated latex from all the clones selected exhibit higher TSC, DRC, particle size and viscosity. Furthermore, during the creaming process, ammonia and potassium laurate soap need to be added in order to prevent coagulation and reduce the viscosity in the latex. It is known that potassium laurate soap is a nonionic surfactant used as stabilizing additives for natural rubber latex. The addition of surfactants during storage modifies the surface of the rubber particles or interface between rubber particles and water by reducing the energy difference between rubber hydrocarbon chains and water. It forms a stable colloidal mixture by reducing the surface tension of all clones selected [28]. Moreover, the addition of creaming agent, ammonia, and surfactant leads to chemical transformations of latex particles along the storage time. Therefore, the amount of non-rubber components or protein should be reduced. The protein content of fresh natural latex is decreased after the creaming process, especially in the case of RRIM600 and PB235 clones reduced the values by 27% and 38%, respectively. Proteins associated with rubber particles are reduced

by strong alkali hydrolysis known as saponification reaction [26,27,29,30]. Finally, the color of rubber according to Lovibond colorimeter in creamed concentrated latex collected from all clones showed lighter color compared to the fresh natural latex, especially the BPM24 clone shown in Figure 7. The removal of some non-rubber constituents from the latex during storage may be the reason for this light color. It was found that the RRIM600 clone exhibits more transparency than other clones. Therefore, it was concluded that the properties of cream concentrated latex depend on creaming agent, stabilizing agent, and non-rubber components.

**Table 6.** TSC, DRC, protein contents, surface tension, viscosity, Lovibond comparator and particles size of the cream concentrated latex from four different *Hevea brasiliensis* clones.

**Figure 6.** Proposed model for the structures of creamed latex with the incorporation of HEC molecules at the surface of rubber particles.

**Figure 7.** The color of cream concentrated latex from four different *Hevea brasiliensis* clones.

#### *3.6. Rubber Particle Size Distribution of Fresh Natural Latex and Cream Concentrated Latex from the RRIM600 Clone*

Figure 8 shows the particle size distribution of fresh natural latex and cream concentrated latex from the RRIM600 clone. The result shows that the diameter of rubber particles in cream concentrated latex are distributed widely in the range of 0.4–15 μm, whereas the fresh natural latex showed a narrower distribution in the range 0.4–5.0 μm. It is clear that the hydroxyethyl cellulose (HEC) molecules covered the surface of rubber particles and might be attributed to all types of rubber particles that can possibly diffuse from the serum into the creamed layers during the creaming process [27]. Thus, the larger size is observed in the rubber particle in the case of concentrated latex. The results are in good agreement with the TEM measurement, as shown in Figure 9. The cream concentrated latex (Figure 9b) showed a greater number of large particles due to the enclosement of HEC and surfactant layer on the surface of particles.

**Figure 8.** Particle size distribution of the fresh natural latex and cream concentrated latex from the RRIM600 clone.

**Figure 9.** Transmission electron micrographs of (**a**) Fresh natural latex; and (**b**) Cream concentrated latex from the RRIM600 clones. (×10,000).

#### *3.7. Comparison between the Mechanical Properties of Fresh Natural Latex and Cream Concentrated Latex fromRRIM600 Clone*

Table 7 shows the green strength, moduli at 100, 300, and 500% elongations, elongation at break, and hardness. Also, Figure 10 shows the stress-strain curve of fresh natural latex, and is compared with the cream concentrated latex. The overall properties of cream concentrated latex were shown to be higher than that of the fresh natural latex, except the elongation at break. This can be explained by the long chain branching in HEC molecules and physical entanglement between HEC and rubber chains. This might be attributed to the crystallization on stretching being related to the number of branch points per chain and chain entanglement [26,31]. This physical entanglement could resist the movement of rubber chains. This result led to the increase in modulus, green strength, and hardness by reducing the elongation at break. Furthermore, the toughness observed from the area under stress-strain curve of the fresh latex exhibit slightly higher than creamed latex. This might be related to the non-rubber components due to the higher non-rubber constituents (proteins and lipids) forming strong structures of loose crosslinks in rubber molecules [10]. Therefore, it is concluded that the green strength is related to the incorporation of highmolecular weight HEC molecules as a creaming agent and non-rubber components, along with rubber molecules.

**Table 7.** Mechanical properties of fresh natural latex and cream concentrated latex from RRIM600 clone.

**Figure 10.** Stress-strain curve of fresh natural latex and cream concentrated latex from RRIM600 clone.

#### **4. Conclusions**

Natural rubber was collected from four different *Hevea brasiliensis* clones (i.e., RRIM600, RRIT251, PB235, and BPM24) and the studies showed a variation in their non-rubber components. The amount of non-rubber components in the latex led to better mechanical and physicochemical properties, such as TSC, DRC, viscosity, surface tension, color, particles size, and molar mass distribution. It was found that the RRIM600, RRIT251, and BPM24 clones showed a protein content of about 3.30 wt%, while the PB235 clone exhibited the least protein content. In addition, the rubber particle size distribution in the RRIM600 clone exhibited a large particle size and a uniform distribution. This resulted in better mechanical properties when compared to the other clones.

Furthermore, the cream concentrated latex was successfully prepared using HEC as a creaming agent and potassium laurate as a creaming aid. It was found that the cream concentrated latex showed higher TSC, DRC, viscosity, particle size, and mechanical properties compared to the fresh natural latex. However, except for the surface tension, protein contents and color were found to be lower than the cream concentrated latex. This is attributed to the incorporation of high-molecular weight HEC molecules and surfactant in the cream concentrated latex. Knowledge acquired from this investigation may lead to various applications according to the unique signature of natural rubber from each clone.

**Author Contributions:** Data curation, writing—original draft preparation, N.L.; Investigation, A.T.; Formal analysis, visualization, L.S.; Methodology, resources, N.U.; Methodology, K.S.; Writing review and editing, J.J.; Visualization, writing—review and editing, Y.N.; Conceptualization, writing review and editing, project administration, funding acquisition, E.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Research Fellowship, Faculty of Science, Prince of Songkla University (contract no. 64001).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This research was also supported by the Research Fellowship, Faculty of Science, Prince of Songkla University (contract no. 64001) and Rubber Product and Innovation Development Research (SCIRU63002), Faculty of Science. We would like to thank Sompong Petrat, Rubber Authority of Thailand for supporting the fresh natural rubber latex.

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

#### **References**

### *Article* **Amination of Non-Functional Polyvinyl Chloride Polymer Using Polyethyleneimine for Removal of Phosphorus from Aqueous Solution**

**Sok Kim 1,2, Yun Hwan Park 1 and Yoon-E Choi 1,\***

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The eutrophication of freshwater environments caused by an excess inflow of phosphorus has become a serious environmental issue because it is a crucial factor for the occurrence of harmful algal blooms (HABs) in essential water resources. The adsorptive removal of phosphorus from discharged phosphorus containing effluents has been recognized as one of the most promising solutions in the prevention of eutrophication. In the present study, a polyvinyl chloride (PVC) polyethyleneimine (PEI) composite fiber (PEI-PVC) was suggested as a stable and recoverable adsorbent for the removal of phosphorus from aqueous phases. The newly introduced amine groups of the PEI-PVC were confirmed by a comparison between the FT-IR and XPS results of the PVC and PEI-PVC. The phosphorus sorption on the PEI-PVC was pH dependent. At the optimum pH for phosphorus adsorption (pH 5), the maximum adsorption capacity of the PEI-PVC fiber was estimated to be 11.2 times higher (19.66 ± 0.82 mg/g) than that of conventional activated carbon (1.75 ± 0.4 mg/g) using the Langmuir isotherm model. The phosphorus adsorption equilibrium of the PEI-PVC was reached within 30 min at pH 5. From the phosphorus-loaded PEI-PVC, 97.4% of the adsorbed amount of phosphorus on the PEI-PVC could be recovered by employing a desorption process using 1M HCl solution without sorbent destruction. The regenerated PEI-PVC through the desorption process maintained a phosphorus sorption capacity almost equal to that of the first use. In addition, consistently with the PVC fiber, the PEI-PVC fiber did not elute any toxic chlorines into the solution during light irradiation. Based on these results, the PEI-PVC fiber can be suggested as a feasible and stable adsorbent for phosphorus removal.

**Keywords:** phosphorus; adsorption; adsorbent; polyvinyl chloride; polyethyleneimine (PEI)

### **1. Introduction**

Phosphorus (P) is widely applied as an important element in various areas of human activity (e.g., agricultural, living, and bio-industrial areas) [1]. Accordingly, a large number of effluents possessing phosphorus have been discharged into water bodies (e.g., rivers and lakes) from various point and non-point routes, such as mining, industrial, sewage, agricultural soil, and surface runoffs [2–4]. Excessively accumulated phosphorus in water bodies can cause eutrophication, which, in turn, causes serious environmental problems such as harmful algal blooms (HABs) in precious water resources because phosphorus is a limiting nutrient for the proliferation of harmful microalgal and cyanobacterial cells [5]. Therefore, the control and removal of phosphorus from aqueous phases is an immensely important task to maintain the quality of water resources.

Various water treatments based on physiochemical and biological technologies, such as electrodialysis, struvite precipitation, membrane filtration, and biological digestion, are being developed and applied to remove phosphorus from aqueous phases [6–10]. Among these methods, chemical precipitation is used to form ammonium phosphate, is

**Citation:** Kim, S.; Park, Y.H.; Choi, Y.-E. Amination of Non-Functional Polyvinyl Chloride Polymer Using Polyethyleneimine for Removal of Phosphorus from Aqueous Solution. *Polymers* **2022**, *14*, 1645. https:// doi.org/10.3390/polym14091645

Academic Editor: Carmine Coluccini

Received: 13 March 2022 Accepted: 18 April 2022 Published: 19 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

recognized as the most well-established phosphorus removal process, and is reported to have the highest removal efficiency (90–95%); however, it can be economically applied to the treatment of an aqueous solution containing a high phosphate concentration (>50 mg/L). Moreover, in the case of biological and membrane processes, some technical limitations have been claimed, such as low effectiveness and strict control requirements (biological process) and high capital and energy cost requirements (membrane process).

Adsorption technology is considered as a reliable and economical application method for the removal of phosphorus from aqueous solutions, because it does not require any additional separate operation processes, additives, or power sources [11]. Therefore, various types of adsorbents have been designed for application in the adsorption process for the removal of phosphorus (phosphate) from aqueous phases. Synthesized metal hydroxide/oxide-based adsorbents, such as Mg(OH)2 and ZrO2, have been reported to have considerable phosphate adsorption capacities at 16.3 and 47.4 mg/g, respectively [12]. Clay minerals, such as palygorskite [13] and clinoptilolite [14], have been investigated as adsorbents for the removal of phosphate from aqueous phases. In addition, biochars derived from wood, corncob, rice husk, and saw dust have gained interested as possible adsorbents for the adsorptive removal of phosphate [15]. However, it is difficult to recover these adsorbents from solution after application due to their small size. When considering phosphorus industrial demands and exhausting natural phosphorus sources, and since phosphorus-loaded adsorbents can be another source of phosphorus, the recovery of adsorbent after use may be important from the viewpoint of industrial sustainability [3]. Molded adsorbents using a polymer matrix have been suggested as the solution for the difficult separation of small, formed adsorbents from solution. For example, chitosan is recognized as a potent polymer matrix of adsorbent for the removal of anionic-formed phosphates by electrostatic attraction due to its content of numerous cationic amine groups and possibility to be immobilized in various hydrogel forms (bead and fibers) [16]. However, since it is a biodegradable biopolymer, it is possible to re-discharge adsorbed phosphorus from chitosan-based adsorbents through natural decomposition of the matrix. Indeed, it has been reported that adsorbed cyanotoxins on chitosan can be released again into the aqueous phase through the degradation of the chitosan matrix [17]. Therefore, to prevent secondary pollution by re-discharging target pollutants, the use of a stable matrix should be considered in the development of molded adsorbents.

In the present study, to fabricate an easily recoverable and highly stable adsorbent for the adsorptive removal of phosphorus, the synthetic polymer polyvinyl chloride (PVC), which is a chemically/physically stable, inexpensive, and light-weight commercial thermoplastic [18], was applied as an adsorbent matrix. Since the adsorption target phosphorus generally appears in anionic phosphate forms (H2PO4 −, HPO4 2−, and PO4 3−) in aqueous phases depending on its pKa property, it can be electrostatically adsorbed to cationic functional groups such as amine groups. However, the chemical structure of PVC does not possess any functional groups as adsorption sites for anionic phosphates. To aminate the PVC matrix, amine-rich ionic polymer polyethyleneimine (PEI) was applied because PEI modification is a well-known method that can efficiently enhance the adsorption performance of adsorbents for anionic pollutants, including phosphates [2,19,20]. Generally, to fabricate PEI-modified molded adsorbents, at least three separate reaction processes are required, including adsorbent molding, PEI-coating/grafting, washing, and cross-linking processes. However, we expected that the PEI-PVC composite adsorbent (PEI-PVC) would only require polymer dissolution and fabrication processes because the amine groups of PEI molecules can be coupled to the alkyl chloride groups of the PVC backbone via the nucleophilic substitution reaction through chloro-groups during the polymer dissolution step [21]. Consequently, we could fabricate the PEI-PVC adsorbent through the direct injection of the dissolving PEI-PVC composite solution into water. In addition, to obtain an easily separable and surface area-maximized PEI-PVC adsorbent, it was fabricated in fiber form. The enhanced amine groups and decreased chloride of the PEI-PVC fiber compared to those of the pristine PVC could be confirmed using FT-IR and XPS analyses. Through pH edge, kinetics, and reuse tests using the PEI-PVC fiber for phosphorus, the pH-dependent, rapid, and reusable phosphorus adsorption properties of the PEI-PVC fiber were determined. The estimated phosphorus adsorption capacity of the PEI-PVC through isotherm tests was comparable to that of other reported adsorbents. Moreover, during utilization as an adsorbent, although the pristine PVC matrix could generate toxic chlorines by photo-oxidative degradation, the PEI-PVC fiber did not elute chlorines under light irradiation conditions. Therefore, the suggested PEI-PVC fiber can be feasible and stable adsorbent for phosphorus adsorption. In addition, our results might provide a sustainable way to valorize waste PVC to a capable adsorbent for the treatment of anionic pollutants.

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

#### *2.1. Materials*

Polyvinyl chloride (PVC, MW of 40 kDa), branched polyethyleneimine (PEI) solution (MW of 750,000, 50% PEI content), and potassium phosphate dibasic (K2HPO4, >98%) were purchased from Sigma-Aldrich Korea Ltd. (Seoul, Korea). *N*,*N*-dimethylformamide (DMF, 99.8%) was supplied by Duksan Science (Seoul, Korea). HCl (36%) and NaOH (>97%) were purchased from Samchun Chemicals Co., Ltd. (Seoul, Korea) and Daejung Chemicals & Metals Co., Ltd. (Siheung, Korea), respectively.

#### *2.2. Preparation of Adsorbents*

To prepare the PEI composite PVC fiber sorbent (PEI-PVC), two grams of PEI solution (50% in solution) was mixed with 30 mL of PVC solution (2 g PVC/30 mL DMF). Next, the mixture was allowed to react at 80 ◦C for 4 h in a water bath. The reacted mixture was continuously injected into a methanol solution (50% *v*/*v* in D.W.) to form a fiber sorbent using a plastic hub needle with air compression. The fabricated PEI-PVC fibers were washed with distilled water several times for the removal of residual DMF and other impurities. The prepared PEI-PVC fiber was then freeze-dried for 24 h. To minimize the effect of humidity and CO2 on the sorbent, it was stored in a desiccator during period adsorption tests.

#### *2.3. Comparison of Functional Group Characteristics in PVC and PEI-PVC Fibers*

The measurement of functional group characteristics on the sorbents was carried out using Fourier transform infrared spectroscopy (FT-IR, Agilent Cary 630 FTIR, Agilent Technology, Santa Clara, CA, USA). FT-IR analyses of adsorbents were conducted within the wavenumber range of 650–4000 cm–1 in the attenuated total reflectance (ATR) mode with scanning number of 100 and 0.9 cm−1 scanning resolution. Furthermore, to analyze minute changes in the functional groups of the sorbents, the XPS signals for N1s and Cl2p of the PVC and PEI-PVC were analyzed via XPS (X-TOOL, ULVAC-PHI, Kanagawa, Japan).

#### *2.4. Evaluation of Phosphorus Adsorption Performance of Adsorbents*

To determine and compare the phosphorus adsorption performances of the PEI-PVC and AC, pH effect (pH edge), kinetics, and isotherm tests were performed in a batch system. In the adsorption experiments, the phosphorus stock solution (initial concentration of phosphorus: 1000 mg/g) was diluted with distilled water to prepare test solutions containing desired initial concentration of phosphorus. For the adsorption test, the adsorbents (adsorbent dosage: 1 g/L) were agitated with the prepared phosphorus solutions in a shaking incubator until reaching the adsorption equilibrium state at 170 rpm and 25 ◦C. In addition, during the adsorption process, the solution pH was continuously monitored and adjusted to the desired pH range using HCl (0.1 M and 1 M) and NaOH (0.1 M and 1 M) solutions. The detailed conditions of each experiment are summarized in Table 1.

#### **Table 1.** Adsorption experimental conditions.

\* To determine the adsorption equilibrium time of adsorbents, during the adsorption experiment, solution samples were taken at different reaction times.

After adsorption processes, to measure residual phosphorus concentration in the experimental samples, liquid–solid separation was conducted using high-speed centrifugation at 9000 rpm. The residual phosphorus concentration in the supernatants was detected using inductively coupled plasma-optical emission spectrometry (ICP-OES, Agilent, Santa Clara, CA, USA). The adsorbed amount of phosphorus by the adsorbents could be calculated using Equation (1) as presented below:

$$q = \frac{V\_i \mathbb{C}\_i - V\_f \mathbb{C}\_f}{M} \tag{1}$$

where *q* (mg/g) is the phosphorus adsorption capacity of the adsorbent. *M* (g) is the applied adsorbent weight to the adsorption experiment. *Ci* and *Cf* are the phosphorus concentrations (mg/L) at the initial and final adsorption states, respectively. *Vi* (L) is the initial volume of the sample, and *Vf* (L) is the final volume considering the amount of added acid/alkaline solution for pH adjustment.

#### *2.5. Determination of PEI-PVC Reusability*

To determine adsorbent reusability of the used PEI-PVC fiber in phosphorus adsorption, phosphorus-loaded PEI-PVC fiber was prepared through adsorption process (agitating 0.03 g of PEI-PVC fiber with 30 mL of phosphorus solution for 4 h at pH 5 and 25 ◦C). The initial phosphorus concentration was 98.25 ± 0.30 mg/L. After the sorption process, phosphorus-loaded PEI-PVC was separated from the mixture and briefly rinsed using 30 mL of distilled water one time to remove residual phosphorus solution. Then, the rinsed phosphorus-loaded PEI-PVC was treated in 30 mL of 1M HCl solution for 4 h to dissociate the adsorbed phosphorus from used PEI-PVC. After that, the regenerated PEI-PVC was washed several times using distilled water to remove the remaining HCl solution. The adsorption process was performed again using completely regenerated PEI-PVC under the same conditions as the first adsorption trial. The adsorption and desorption processes using PEI-PVC were repeated in three cycles.

#### *2.6. Measurement of Chlorine Elution from PVC-Based Sorbents*

To measure the eluted chlorine levels that were generated from the PVC backbone of the sorbents, PVC and PEI-PVC fibers were agitated in 150 mL of autoclaved distilled water for 24 h under white fluorescent light (50 mol/m2) at 25 ◦C. The eluted chlorine concentration in the gathered samples was measured using a residual chlorine meter (HI 96710, Hanna, Woonsocket, RI, USA) following the manufacturer's protocol. All experiments were conducted thrice.

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

#### *3.1. Change of Functional Group Properties of PVC Sorbent after PEI Reaction*

Since the adsorption target phosphorus usually exhibits anionic phosphate forms in an aqueous solution depending on its pKa properties, cationic functional groups possessing sorbents can adsorb phosphates by electrostatic attraction. Therefore, the determination of the existing cationic binding sites on the sorbents might be an important factor to confirm the applicability of adsorbents to phosphorus adsorption processes. In the present study, the newly generated cationic functional groups of the PEI-PVC fiber by the PEI and PVC composite were analyzed using FT-IR and XPS instruments. Figure 1 presents the FT-IR spectrum of the PVC and PEI-PVC. As shown in Figure 1, the PEI-PVC fiber showed FT-IR peaks associated with the base material PVC. The peaks in the range of 2921–2852 cm−1 correspond to the symmetric and symmetrical stretching bonds of C–H in the PVC molecules [22,23]. It has been reported that peaks at 2965 and 1245 cm−1 are related to the C–H stretching of CH–Cl in the PVC molecule [24]. In addition, the peaks at 1430 (C–H bending of –CH2), 1324 (C–H deforming in plane), 1093 (C–C stretching), and 970 cm−1 (CH2 rocking) indicate the chemical bond properties of the PVC matrix [25]. In the FT-IR spectra of the PEI-PVC, FT-IR adsorption peaks associated with amine groups could be found, whereas those of the PVC did not show. The newly observed broad peak in the range of 3600–3200 cm−1 and the strong peak at 1654 cm−1 are attributed to the asymmetric N–H stretching of amines and the bending of secondary amines, respectively [26]. In addition, the appearance of the small peak at 1440 cm−1 can be attributed to –NH bending or C–N stretching [26,27]. Furthermore, the intensity of the peak at 1245 cm−1 assigned to the bending bond of C–H near Cl in the PVC backbone decreased and shifted to 1235 cm−1 in the FT-IR spectra of the PEI-PVC. These observed peak changes in the FT-IR result of the PEI-PVC fiber might indicate increased amine groups and decreased Cl amounts in the PEI-PVC fiber due to a nucleophilic substitute reaction between the chloro-groups of the PVC backbone and the amine groups of the PEI molecules [21]. Additional evidence related to these changes in functional groups could be confirmed by XPS analyses for nitrogen (XPS N1s) and chloride (XPS Cl2p) atoms of the PVC-based fibers (Figure 2). When comparing the XPS N1s signals between the PVC and PEI-PVC fibers, the PVC fiber did not show any XPS signals related to the nitrogen-based chemical bonds (i.e., amine groups). However, in the case of the PEI-PVC, the strong N1s signal was newly observed in the binding energy range of 392–404 eV. This signal might be attributed to the amine groups of the coupled PEI molecules with the PVC backbone since N1s peaks of –NH2 and C–N = C bonds in the PEI molecules could be observed at 398.8 and 399.5 eV [19]. This result is clearly connected to the newly appeared FT-IR peaks of the amine groups in the FT-IR spectra of the PEI-PVC fiber. In the case of XPS Cl2p signals, a higher XPS signal intensity was observed in the XPS result of the PVC than in that of the PEI-PVC. This evidently indicates that the amount of chloride was decreased in the PEI-PVC fiber compared to that of the PVC fiber owing to the substitution reaction between the PEI molecules and the chloride in the PVC.

The decreased amount of chloride in the PEI-PVC compared to the pristine PVC could be additionally supported by the different eluted chlorine concentrations from the PVC and PEI-PVC fibers under light irradiation conditions. It has been reported that PVC can generate toxic chlorines via the photo-degradation of the backbone structure [28]. As shown in the XPS Cl2p results, since the amount of chloride in the PEI-PVC was reduced, it was expected that the elution of chlorines from the PEI-PVC fiber might be reduced compared to that of the PVC. Therefore, the generated chlorines from the PVC and PEI-PVC fibers were compared (Figure 3). As we hypothesized, the pristine PVC fiber released 0.12 ± 0.002 mg/L of chlorine into the aqueous solution during light exposure for 24 h, whereas the PEI-PVC fiber did not. The PEI-PVC-agitated sample showed a chlorine concentration almost the same as that of the control water (0.013 ± 0.001 mg/L). Furthermore, the prevention of toxic chlorine emissions from the PEI-PVC fiber under light conditions might be a meaningful result indicating that the PEI-PVC fiber is directly applicable and a stable adsorbent for the removal of phosphorus in actual environments.

**Figure 1.** FT-IR spectrum of PVC and PEI-PVC.

**Figure 2.** XPS N1s and Cl2p signals of PVC-based sorbent materials.

**Figure 3.** Eluted chlorine levels from PVC and PEI-PVC in an aqueous solution.

#### *3.2. pH Effect on Phosphorus Adsorption*

Through the FT-IR and XPS measurements, it was determined that amine groups derived from PEI molecules are the main binding sites of the PEI-PVC fiber, which can electrostatically adsorb phosphates. Since the electrostatic attraction between the PEI-PVC fiber and phosphates can be affected by the solution pH due to the deprotonation of the amine groups of the PEI-PVC and the phosphates in the solution depending on their pKa properties, the solution pH condition is an important factor in the adsorption process [2]. In the present study, the pH effect on the phosphorus adsorption of the PEI-PVC was evaluated under different solution pH conditions (pH 2–8). In addition, the pH effect on the phosphorus sorption capacity of the conventional adsorbent activated charcoal (AC) was evaluated to make a comparison with that of the PEI-PVC fiber.

Figure 4 illustrates the pH effect on the phosphorus uptakes of the PEI-PVC and AC. As displayed in Figure 4, the PEI-PVC exhibited a pH-dependent phosphorus adsorption property. With an increase in solution pH from 2 to 3.5, the phosphorus sorption capacity of the PEI-PVC increased accordingly. At solution pH 3.5, the phosphorus sorption capacity of the PEI-PVC was recorded as approximately 27 mg/g (26.8 ± 0.48 mg/g), and it showed similar phosphorus sorption capacities until pH 5. Above pH 5, the phosphorus uptake of the PEI-PVC fiber decreased substantially. The pH-dependent phosphorus adsorption of the PEI-PVC fiber can be explained by the electrostatic interaction between the amine groups of the PEI-PVC and phosphate anions, as we assumed previously. It has been reported that the pKa values of amine groups (e.g., primary, secondary, and tertiary amine groups) in PEI molecules can be observed at approximately pH 4.5, 6.7, and 11.6, respectively [29]. Accordingly, the amine groups on the PEI-PVC derived from PEI molecules can be fully positively charged under acidic conditions below pH 4; therefore, it was expected that the phosphorus sorption uptake of the PEI-PVC might record the maximum value at pH 2. However, the phosphorus sorption uptake of the PEI-PVC at pH 2 was significantly lower than that at pH 3.5. This result might be attributed to the pH dependence of phosphate speciation. Phosphorus can exist as uncharged phosphoric acid (H3PO4) and electrostatically adsorbable phosphate anions (H2PO4 −, HPO4 2−, and PO4 3−) depending on the solution pH condition in the aqueous solution [30]. According to our previous study [2], only 40% of total phosphorus amounts can be presented as the electrostatically adsorbable phosphate anion H2PO4 − at pH 2. However, the almost amount of phosphorus

can be changed to the phosphate anionic form at pH 3.5. The increasing fraction of anionic phosphate might contribute to the increasing phosphorus uptake of the PEI-PVC in the range of pH 2–pH 3.5 by the enhancement of the electrostatic attraction between the PEI-PVC and phosphate. Above pH 5, although the almost amount of phosphorus can be formed as the adsorbable phosphate anions, since the positivity of the PEI-PVC could be lost by the deprotonation of amine groups depending on their pKa properties, the phosphorus sorption capacity of the PEI-PVC might be reduced. The conventional adsorbent, AC, did not show pH dependence on phosphorus adsorption capacity. However, the phosphorus sorption capacities of the AC were significantly lower than those of the PEI-PVC fiber under all of the pH conditions.

**Figure 4.** pH effect on the phosphorus sorption uptake of PEI-PVC and activated charcoal.

#### *3.3. Reusability of PEI-PVC Fiber*

The pH-dependence property of the PEI-PVC fiber on phosphorus adsorption could be applied to the phosphorus desorption process for the reuse of the PEI-PVC. As discussed above, under highly acidic conditions, since phosphate anions can be changed to uncharged phosphoric acid (H3PO4), adsorbed phosphorus might be recovered from phosphate-loaded PEI-PVC. To estimate the desorbed amount of phosphorus, the phosphate-adsorbed PEI-PVC at pH 5 was treated using 1M HCl solution. Figure 5 displays the measured amounts of the adsorbed and desorbed phosphorus through the sorption and desorption processes using the PEI-PVC fiber. As shown in Figure 5, 1.93 ± 0.01 mg of phosphorus could be adsorbed on the PEI-PVC at pH 5 with the first use of the PEI-PVC fiber. In addition, after the treatment of the phosphate-loaded PEI-PVC using the 1M HCl solution, the almost amount of adsorbed phosphorus on the PEI-PVC fiber could be desorbed (1.88 ± 0.05 mg) owing to the loss of electrostatic attraction between the binding site and the sorbate through the speciation change from phosphate anions to uncharged phosphoric acid under highly acidic conditions. From the replicated adsorption and desorption tests, it was found that the regenerated PEI-PVC could adsorb a similar amount of phosphorus to that used in the first trial. In the second and third trials of phosphorus adsorption using regenerated PEI-PVC, 1.78 ± 0.06 and 1.84 ± 0.04 mg of phosphorus were adsorbed on the regenerated PEI-PVC, respectively. When considering the reusability of PEI-PVC, the adsorptive removal process of phosphorus using PEI-PVC might have an economic benefit.

**Figure 5.** Reusability of PEI-PVC in three cycle applications (adsorbed phosphorus at pH 5 and desorbed in 1 M HCl solution).

#### *3.4. Adsorption Kinetics*

Adsorption equilibrium time is an important factor to determine the efficiency of the developed sorbent for the target materials. Therefore, to determine the sorption equilibrium time of the PEI-PVC for phosphorus, a kinetic experiment using the PEI-PVC was performed in a batch system at pH 5. From the kinetic result (Figure 6), the PEI-PVC showed a rapid adsorption equilibrium at approximately 30 min for phosphate anions at pH 5. In the case of the AC, a faster phosphorus adsorption equilibrium was attained than that attained with the PEI-PVC (within 15 min). However, this might be attributable to the significantly low phosphorus sorption capacity of the AC compared to that of the PEI-PVC. To compare the phosphorus sorption kinetic parameters of the PEI-PVC and AC in detail, a pseudo-second-order kinetic model was applied to the experimental kinetic data of the PEI-PVC fiber and AC because it is one of the representative kinetic models used to determine the kinetic properties of adsorbents. The pseudo-second-order kinetic model is represented as Equation (2):

$$q\_t = \frac{q\_c^2 k\_2 t}{1 + q\_c k\_2 t} \tag{2}$$

where *qe* and *qt* are the phosphorus sorption capacity of the sorbents (mg/g) at the sorption equilibrium state and specific sorption reaction time (*t*), respectively. The parameter *k*2 (g/mg min) indicates the estimated adsorption rate constants from the pseudo-secondorder model, respectively.

**Figure 6.** Phosphorus sorption kinetics of PEI-PVC and activated charcoal at pH 5.

The estimated kinetic parameters for the PEI-PVC and AC from the pseudo-secondorder kinetic model are summarized in Table 2. According to Table 2, the estimated correlation coefficients (R2) of the pseudo-second-order model for the phosphorus sorption kinetic data of the PEI-PVC and AC were 0.9585 and 0.9211, respectively. These high R2 values indicate that the phosphorus sorption kinetic experimental results of the PEI-PVC and AC were predicted well by the pseudo-second-order kinetic model. The sorption reaction rate (k2) of the AC was estimated as 0.037 ± 0.013 g/mg·min using the kinetic model. This was a value approximately 6.5 times higher than that of the PEI-PVC (0.0057 ± 0.0007 g/mg·min), and it was better connected to the faster sorption equilibrium state of the AC than the PEI-PVC fiber. However, as shown in the experimental kinetic results, the PEI-PVC showed a comparable sorption equilibrium time to that of the AC. Furthermore, the phosphorus sorption capacity (qe) calculated from the sorption kinetic data of the PEI-PVC was 5.9 times higher (23.39 ± 0.47 mg/g) than that of the AC (3.94 ± 0.21 mg/g) using the pseudo-second-order model. Based on the kinetics result, the PEI-PVC fiber can be suggested as an efficient substitute for conventional adsorbents (i.e., activated carbons) to rapidly counteract the inflow of phosphorus from the effluents of point or non-point pollutant sources.

**Table 2.** Adsorption kinetic parameters of applied sorbents for phosphorus estimated from pseudosecond-order model at pH 5.

#### *3.5. Maximum Phosphorus Sorption Capacity of PEI-PVC Fiber*

Together with the sorption kinetic property, the maximum sorption capacity (qmax) of the sorbent toward the target pollutant is an essential factor for the evaluation of the adsorption performance of the developed adsorbent. Therefore, to determine the qmax of the developed sorbent PEI-PVC fiber, isotherm experiments were performed under various pH conditions (pH 5–7). In addition, the maximum phosphorus (i.e., phosphate anions) sorption capacities of the PEI-PVC fiber were compared with those of the conventional adsorbent (AC). The obtained isotherm results using the PEI-PVC and AC at different pH values are presented in Figure 7. As shown in Figure 7, the phosphorus sorption capacities of the PEI-PVC and AC showed increasing values as the equilibrium phosphorus concentration was increased. In addition, above the specific equilibrium phosphorus concentration, the phosphorus adsorption uptakes of the PEI-PVC and AC reached the adsorption equilibrium state at all pH values. The phosphorus adsorption uptakes of the PEI-PVC fiber at the equilibrium state decreased from approximately 20 mg/g to 13 mg/g as the solution pH was increased from pH 5 to pH 7. The AC showed almost constant phosphorus adsorption capacities (average 1.75 ± 0.4 mg/g) at the adsorption equilibrium state regardless of pH conditions. These phosphorus uptakes of the AC were clearly low compared to those of the PEI-PVC fiber. Therefore, extensively used isotherm models (Freundlich and Langmuir isotherm model equations) were applied to the only experimental isotherm results of the PEI-PVC for the estimation of detailed isotherm parameters. The Freundlich and Langmuir isotherm model equations are presented as Equations (3) and (4), respectively. The estimated isotherm parameters from these model equations are presented in Table 3.

$$q\_{\mathfrak{e}} = \mathbb{K}\_{\mathfrak{f}} \mathbb{C}\_{\mathfrak{f}}^{\frac{1}{n}} \tag{3}$$

$$q\_{\varepsilon} = \frac{q\_{\max, \, L} b \mathbb{C}\_f}{1 + b \mathbb{C}\_f} \tag{4}$$

where *qe* is the experimentally calculated phosphorus sorption capacity (mg/g). *Cf* (mg/L) is the final phosphorus concentration in the solution after the adsorption process. The parameters *Kf* (L/g) and *n* are the Freundlich constants, which can be indicators of sorption capacity and sorption intensity, respectively. The parameters in the Langmuir model equation *qmax,L* and *b* indicate the maximum capacity (mg/g) and sorption affinity (L/mg) of the adsorbent for phosphorus, respectively.

As summarized in Table 3, the Freundlich parameter (*Kf*) associated with the phosphorus adsorption capacity of the PEI-PVC fiber increased with the decrease in the solution pH from pH 7 (5.12 ± 0.97 L/g) to pH 5 (9.83 ± 1.34 L/g). The dimensionless n value of the PEI-PVC in phosphorus adsorption showed a similar tendency to that of the *Kf* values. The recorded n values from the isotherm data of the PEI-PVC fiber for phosphorus at pH 5, pH 6, and pH 7 were 5.21 ± 1.05, 5.29 ± 1.10, and 4.14 ± 0.91, respectively. According to Toor and Jin [31], if the n value is larger than 1, it can be determined that the adsorption process is favorable. Therefore, the phosphorus adsorption process with application of the PEI-PVC fiber might be a favorable process. The application of the Langmuir isotherm model to the experimental isotherm data of the PEI-PVC showed better fitting lines, because of its higher determination coefficients (R2 values: 0.9755, 0.9843, and 0.9844 at pH 5, 6, and 7, respectively), than those of the Freundlich model (R2 values: 0.9192, 9161, and 0.9046 at pH 5, 6, and 7, respectively) at all solution pH values. The maximum phosphorus sorption capacities (*qmax,L*) of the PEI-PVC fiber were estimated from the Langmuir model as 19.66 ± 0.82, 16.54 ± 0.54, and 13.66 ± 0.51 mg/g at pH 5, 6, and 7, respectively. In addition, the isotherm result of the PEI-PVC fiber showed a higher *b* value at pH 5 (2.08 ± 0.59 L/mg) than at pH 6 (1.65 ± 0.35 L/mg) and pH 7 (0.42 ± 0.08 L/mg) using the Langmuir model. As discussed above section, since the higher amount of amine groups in the PEI-PVC fiber could be positively activated at pH 5 compared to those at pH 6 and pH 7 depending on the pKa properties of the amine groups, the higher values of the Freundlich and Langmuir isotherm parameters corresponding to the sorption capacity and affinity for phosphorus adsorption could be calculated from the isotherm data.

**Figure 7.** Isotherm results of PEI-PVC and activated charcoal for phosphorus adsorption at pH 5, 6, and 7.

**Table 3.** Estimated isotherm parameters of PEI-PVC for phosphorus from Freundlich and Langmuir models.

Furthermore, a comparison of the maximum phosphorus adsorption capacity of the PEI-PVC fiber with that of previously reported adsorbents was performed (Table 4). As shown in Table 4, in general, the adsorbents containing Zr showed higher *qmax* values than those of the PEI-PVC fiber at natural pH. For instance, the qmax values for the phosphorus of Zr-loaded orange waste gel, Zr-loaded MUROMAC, and Zr-loaded okara were calculated as 57, 43, and 14.39 mg/g, respectively. Under the condition of pH 3, the covalently cross-linked chitosan by epichlorohydrin showed higher phosphorus adsorption performance (38.22 mg/g) compared to the PEI-PVC fiber (approximately 27 mg/g), whereas the maximum phosphorus uptake of electrostatically cross-linked chitosan by sodium citrate (13.3 mg/g) was smaller than that of the PEI-PVC. Although we could find several adsorbents showing higher qmax values for phosphorus than the PEI-PVC fiber, there still remains an opportunity to enhance the phosphorus adsorption performance of the PEI-PVC fiber through the optimization of adsorbent processes and the additional application of adsorption-supporting materials. In addition, as shown in Table 4, the PEI-PVC fiber still displayed superior or comparable phosphorus adsorption performance to other previously reported sorbents. For example, zirconium ferrite [32], Fe-loaded juniper fiber [33], iron–hydroxided eggshell [34], and La(III)-modified fine needle [35] showed maximum phosphorus sorption capacities of 13, 2.31, 14.49, and 6.31 mg/g at pH 7, respectively.

**Table 4.** Maximum phosphorus adsorption capacities of previously reported adsorbents.

#### **4. Conclusions**

In the present study, the amination of a non-functional PVC matrix was performed using a simple chemical reaction process between PEI and PVC to develop an adsorbent for phosphorus removal from aqueous phases. From the FT-IR and XPS-N1s results of the prepared PEI-PVC, the aminated characteristics of the PEI-PVC fiber were well determined. The adsorption of phosphorus on the PEI-PVC was affected by the solution pH. The maximum phosphorus sorption capacity (qmax) of the PEI-PVC fiber was estimated as 19.66 ± 0.82 mg/g at pH 5 using the Langmuir equation. At pH 6 and pH 7, the qmax values of the PEI-PVC fiber were calculated as 16.54 ± 0.54 and 13.66 ± 0.51, respectively. In the kinetic analysis, the PEI-PVC showed a rapid phosphorus adsorption equilibrium time within 30 min at pH 5. The used PEI-PVC could be regenerated without destructing the adsorbent matrix using the 1M HCl solution by the desorption of the adsorbed phosphorus in the PEI-PVC fiber (desorption rate: >98%). In addition, the regenerated PEI-PVC was reusable for phosphorus adsorption without the loss of sorption capacity. Furthermore, it was found that the PEI-PVC fiber did not discharge toxic chlorines during light irradiation. Based on the results, we can suggest that the stable and reusable PEI-PVC fiber can be a feasible adsorbent for phosphorus removal from aqueous phases. However, to further enhance its phosphorus adsorption performance, additional research on the optimization of adsorbent processes and the additional application of adsorption-supporting materials might be needed.

**Author Contributions:** Conceptualization, S.K.; methodology, S.K. and Y.H.P.; validation, S.K. and Y.- E.C.; formal analysis, S.K.; investigation, S.K.; resources, S.K. and Y.H.P.; data curation, S.K.; writing original draft preparation, S.K. and Y.H.P.; review and editing, S.K. and Y.-E.C.; visualization, Y.H.P. and S.K.; supervision, Y.-E.C.; project administration, Y.-E.C. and S.K.; funding acquisition, Y.-E.C. and S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Korea Environment Industry & Technology Institute (KEITI) through a project to develop eco-friendly new materials and processing technology derived from wildlife, funded by the Korea Ministry of Environment (MOE)<PHONE_NUMBER>004). This research was also financially supported by the National Research Foundation of Korea (NRF) (grants NRF-2021R1I1A1A01054658 and NRF-2021R1A6A1A10045235).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data of manuscript is an original research work and has not been published elsewhere.

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

#### **References**

### *Article* **Rheological and Thermal Study about the Gelatinization of Different Starches (Potato, Wheat and Waxy) in Blend with Cellulose Nanocrystals**

**Josefina Chipón 1, Kassandra Ramírez 1, José Morales 2,3 and Paulo Díaz-Calderón 2,3,\***

**Abstract:** The goal of this work was to analyze the effect of CNCs on the gelatinization of different starches (potato, wheat and waxy maize) through the characterization of the rheological and thermal properties of starch–CNC blends. CNCs were blended with different starches, adding CNCs at concentrations of 0, 2, 6 and 10% *w/w*. Starch–CNC blends were processed by rapid visco-analysis (RVA) and cooled to 70 ◦C. Pasting parameters such as pasting temperature, peak, hold and breakdown viscosity were assessed. After RVA testing, starch–CNC blends were immediately analyzed by rotational and dynamic rheology at 70 ◦C. Gelatinization temperature and enthalpy were assessed by differential scanning calorimetry. Our results suggest that CNCs modify the starch gelatinization but that this behavior depends on the starch origin. In potato starch, CNCs promoted a less organized structure after gelatinization which would allow a higher interaction amylose–CNC. However, this behavior was not observed in wheat and waxy maize starch. Insights focusing on the role of CNC on gelatinization yielded relevant information for better understanding the structural changes that take place on starch during storage, which are closely related with starch retrogradation. This insight can be used as an input for the tailored design of novel materials oriented towards different technological applications.

**Keywords:** gelatinization; cellulose nanocrystals; rheology; calorimetry

#### **1. Introduction**

Starch and cellulose are the most widely distributed polymers in nature. Starch is found in the form of granules which are energy reservoirs for plants [1]. Cellulose, in turn, is part of the structural basis of cell walls in plant tissues, normally forming complexes with hemicellulose and lignin [2]. Although starch and cellulose have been widely used in food science and technology, in recent years, the interest in exploring the novel applications of these polymers and their composites has increased across completely different fields. Indeed, the literature has reported the use of cellulose as a strategy to improve some poor physical properties of starch such as brittleness, low mechanical resistance, high gas permeability and high hygroscopicity [3,4]. This strategy can be useful for the tailored design of starch–cellulose composites destined for food packaging and coating materials [4–7], scaffolds for tissue engineering (e.g., wound healing) [8–10] as well as applications in bioengineering and the pharmaceutical industry [3,8,11].

Likewise, more recently, interest in exploring the use of nanocellulose in the design of starch-based composites has also increased. Among the more interesting choices are

**Citation:** Chipón, J.; Ramírez, K.; Morales, J.; Díaz-Calderón, P. Rheological and Thermal Study about the Gelatinization of Different Starches (Potato, Wheat and Waxy) in Blend with Cellulose Nanocrystals. *Polymers* **2022**, *14*, 1560. https:// doi.org/10.3390/polym14081560

Academic Editors: Valentina Siracusa, Nadia Lotti, Michelina Soccio and Alexey Iordanskii

Received: 17 March 2022 Accepted: 10 April 2022 Published: 11 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

cellulose nanocrystals (CNCs). CNCs correspond to the crystalline fractions presented in the elemental fiber of a cellulose microfibril [12,13]. CNCs are normally produced by acid hydrolysis under severe conditions in terms of concentration and temperature, which allows obtaining nanosized crystalline whiskers [14,15]. Depending on hydrolysis conditions and the cellulose source, CNCs have dimensions of approximately 120–200 nm in length and have a negative charge (z-potential lower than −30 mV) [14,15].

Despite the effect of CNCs on the physical properties and functionality of different starches being described in the literature, studies describing the role of CNCs during the gelatinization of starch are scarce. The gelatinization of starch has been well-described in terms of the mechanism of granule swelling, granule disruption, the loss of birefringence and amylose and amylopectin leaching due to the effect of temperature and stirring [16–18]. However, a comprehensive study of the gelatinization of starch in the presence of nanocrystals will help to understand the behavior of starch during the storage when the complex process of self-association and self-assembling will take place. This behavior explains the retrogradation of starch.

Therefore, this work aimed to study the gelatinization of starches from different sources (potato, wheat and waxy maize) in the presence of CNCs produced from cotton cellulose pulp. Pasting parameters closely related to starch gelatinization (such as pasting temperature, peak viscosity, hold viscosity and breakdown viscosity) were assessed in starch–CNC blends by rapid visco-analysis (RVA). The viscoelasticity of these blends were evaluated by rheology test at 70 ◦C, whereas the gelatinization temperature and enthalpy were assessed by differential scanning calorimetry (DSC).

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

#### *2.1. Materials*

Native potato (Avebe, Veendam, The Netherlands), wheat (Sigma Aldrich, Darmstadt, Germany) and waxy maize (Sigma Aldrich, Darmstadt, Germany) starches were purchased in powder form. Cellulose nanocrystals (CNCs) produced from cotton fibers were purchased in freeze-dried form from The Process Development Center of University of Maine (Orono, ME, USA). Starch and CNCs were used as received without further purification and stored at room temperature until further use.

#### *2.2. Methods*

#### 2.2.1. Sample Preparation

CNC suspensions were prepared for blending with native starch to have a starch suspension with an CNC concentration of 0, 2, 6 and 10% *w/w* dry cellulose (Equation (1)). For this purpose, well-defined amounts of CNCs were suspended in distilled water (conductivity < 10 mS) only by stirring. Before being mixed with starch, CNCs were sonicated (1500 W, 5 min) at ambient temperature (20 ± 2 ◦C) to promote the complete dispersion of nanocrystals:

$$\text{CNC concentration } (\%, w/w) = \text{(CNC}\_{\text{weight}} / (\text{CNC}\_{\text{weight}} + \text{Starth}\_{\text{weight}})) \times 100 \tag{1}$$

#### 2.2.2. Gelatinization of Starch–CNC Blends by Rapid Visco-Analysis (RVA)

The gelatinization of starch–CNC blends was carried out by rapid visco-analysis (RVA 4500, Perten Instruments, North Ryde, Australia) in accordance with the methodology proposed by Diaz-Calderon et al. [19] with minor modifications. Two grams of native starch was weighed in aluminum canisters and 25 mL of CNC suspension with different cellulose concentration was transferred using a micropipette. The canister was then inserted into the instrument and viscosity patterns were obtained as a function of temperature: holding at 25 ◦C during 2 min; heating between 25 and 95 ◦C at 13.5 ◦C/min; holding at 95 ◦C for 5 min; cooling to 25 ◦C at 13.5 ◦C/min; and holding at 25 ◦C for 1 min. The analysis was performed under constant stirring (160 rpm). As the goal of this work was focused on understanding the gelatinization of native starch, the pasting parameters evaluated were pasting temperature (◦C), peak viscosity (cP), hold viscosity (cP) and breakdown viscosity (difference between hold viscosity and peak viscosity, cP). All measurements were carried out at least in triplicate.

#### 2.2.3. Rheological Characterization of Gelatinized Starch–CNC Blends

The changes in viscoelasticity of starch–CNC blends were carried out by rheology (Discovery HR-2, TA Instrument, New Castle, DE, USA). Samples analyzed by rheology were prepared using the RVA machine and following the same protocol explained in Section 2.2.2, but with the cooling stage finishing at 70 ◦C. Once the protocol was finished, the RVA canister was immediately transferred to a controlled bath set at 70 ◦C. Samples were collected from the canister and transferred to the rheometer.

Apparent viscosity (Pa·s) and shear stress (Pa) were assessed from the rotational flow curve obtained at 70 ◦C using cone geometry (stainless steel, 40 mm diameter, 0:30:7 angle and 15 μm truncation) in the shear rate range between 1 and 1000 1/s.

Dynamic changes in storage modulus (G , Pa) and loss modulus (G", Pa) were obtained through a frequency sweep carried out at 70 ◦C to analyze the behavior of G , G", and the loss factor (G"/G ) as a function of angular frequency from 1 to 648 rad/s at 0.5% strain which was within the linear viscoelastic range (LVR) previously defined by an amplitude sweep (0.1–1000%) at 70 ◦C. A flat plate geometry (5 cm diameter) was used for analysis and a 300-micron gap was selected for testing. The analysis considered at least five replicates per experimental condition.

#### 2.2.4. Thermal Properties of Gelatinized Starch–CNC Blends

Gelatinization temperature (◦C) and enthalpy (J/g starch) were measured by differential scanning calorimetry (DSC 1, Mettler-Toledo, Greinfensee, Switzerland) following the protocol reported by Díaz-Calderón et al. [19]. In order to improve the resolution signal, a higher concentration of the starch weight suspensions was used, keeping the CNC weight previously indicated fractions (0–10% *w/w*). The starch concentration was 20% *w/v*. Approximately 60 μL of starch–CNC blend suspensions were loaded into 100 μL aluminum pans and then hermetically sealed. The DSC was calibrated using indium (melting temperature and enthalpy of 156.5 ± 1.56 ◦C, H = 28.6 ± 1 J/g), and an empty pan was used as a reference. Thermal properties of the suspensions were measured as follows: holding temperature at 5 ◦C during 3 min, heating from 5 ◦C to 85 ◦C at 10 ◦C/min, and holding at 85 ◦C during 3 min. The gelatinization temperature (◦C) was recorded from the onset of the endothermic peak associated with the starch granule swelling, while gelatinization enthalpy was considered as the area under the endothermic peak. Gelatinization enthalpy was normalized in terms of starch dry mass and was expressed in J/g starch. All measurements were performed at least in triplicate.

#### 2.2.5. Statistical Analysis

Where appropriate, the statistical significance was assessed by a paired *t*-test (same variances) and ANOVA using the Solver tool in Excel (Office 2016, Microsoft Corp., Redmond, WA, USA).

#### **3. Results**

#### *3.1. Gelatinization of Starch–CNC Blends by Rapid Visco-Analysis (RVA)*

Viscosity patterns of starch samples resulted from RVA are presented in Figure 1. As expected, the viscosity pattern showed marked differences among the starches tested: potato, wheat and waxy maize. From the viscosity patterns, the pasting parameters that allowed characterizing the gelatinization of starch were assessed, which in our study were pasting temperature, peak viscosity, hold viscosity and breakdown viscosity. The assessments of the pasting parameters for our starch–CNC blends are presented in Table 1.

Significant differences (*p*-value < 0.05) in the pasting parameters were observed among the pure starch samples (0%CNC). Regarding the pasting parameters of starch–CNC blends, our results show that the presence of CNC did not produce a significant difference (*p*-value > 0.05) on the pasting temperature. However, CNC produced a significant decrease (*p*-value < 0.05) in peak viscosity which was proportional to the increase in CNC concentration only in potato starch. In wheat and waxy starch, a decrease in peak viscosity was only detected at the highest CNC concentration tested. On the other hand, hold viscosity (also called through viscosity or hot-paste viscosity [16]) was significant lower (*p*-value < 0.05) only at the highest CNC concentration tested (10%) in potato and waxy maize starch. This parameter was not significant modified (*p*-value > 0.05) in the wheat starch blend. Likewise, the breakdown viscosity was significantly reduced (*p*-value < 0.05) by CNC in potato starch, and only at the highest CNC level tested in wheat and waxy starch. Interestingly, the assessment of the relative decrease only showed a marked decrease in potato starch, whereas in wheat and waxy starch, this ratio was lower only at the highest CNC level tested.

**Figure 1.** RVA profiles in starch from the different sources: potato (orange line), wheat (grey line) and waxy maize (blue line). Dotted line represents the temperature profile used during analysis.

#### *3.2. Rheological Characterization of Gelatinized Starch–CNC Blends*

Rheology analysis by rotational and dynamic tests were carried out at 70 ◦C on samples completely gelatinized by RVA. Apparent viscosity and shear stress as a function of shear rate recorded in pure starch samples and starch–CNC blends are presented in Figures 2 and 3, respectively. In agreement with the hold viscosity data assessed by RVA (Table 1), the flow curve showed significantly higher viscosity values in potato starch at 70 ◦C over the complete shear rate range tested, whereas the waxy starch showed the lowest viscosity at 70 ◦C. Independent of the source, all starches showed non-Newtonian behavior.

Figure 3 shows the effect of different concentrations of CNC on the apparent viscosity and shear stress as a function of shear rate tested at 70 ◦C in complete gelatinized starches. Our results show that CNC produced only slight changes in apparent viscosity and shear stress. Indeed, well-defined viscosity data recorded at 40 1/s (Table 2) showed that the presence of CNC only significant modified (*p*-value < 0.05) the apparent viscosity of waxy starch at 6% and 10%, tested at 70 ◦C. Likewise, the non-Newtonian condition of gelatinized starches tested at 70 ◦C was not modified by the presence of CNC, independently of the starch source. As expected, and following the same trend observed in Figure 3, gelatinized potato starch–CNC blends showed the highest apparent viscosity values, whereas waxy maize–CNC showed the lowest values of viscosity, independently of the CNC concentration. The same behavior was observed in terms of shear stress as a function of shear rate in the starch–CNC blends.

**Figure 2.** Flow curve in gelatinized pure starch samples (0%CNC) at 70 ◦C: (**a**) apparent viscosity and (**b**) shear stress as a function of shear rate.

**Figure 3.** Flow curve in gelatinized starch samples at 70 ◦C and at different concentrations of CNCs: (**a**) potato; (**b**) wheat; and (**c**) waxy maize. Top plots correspond to apparent viscosity as a function of shear rate, whereas bottom plots correspond to shear stress as a function of shear rate.

The viscoelastic characterization of starch–CNC blends is presented in Figures 4 and 5. Dynamic tests were carried out within the linear viscoelastic range previously defined by an amplitude sweep at 70 ◦C (Figures S1 and S2, Supplementary File). Wheat and waxy starch did not show significant differences (*p*-value > 0.05) in G and both showed a slight slope over the angular frequency range tested at 70 ◦C (Figure 4a). Potato starch showed a significantly lower G over the whole angular frequency tested along with a marked slope depicting the strong dependence of G with the angular frequency. All the gelatinized starch samples showed significantly higher values of G compared to values of G", which represent the gel-like condition of starch blends after processing by RVA. The latter is also confirmed from the values of the loss factor at 70 ◦C which resulted in values lower than 1 in all samples tested (Figure 4b). However, at angular frequencies lower than 100 rad/s, the loss factor in potato starch was constant and with higher values than those assessed in the wheat and waxy starch.

**Table 2.** Apparent viscosity and shear rate of starch–CNC blends assessed at a shear rate of 40 1/s and 70 ◦C. Values in brackets correspond to standard deviation. Different upper letters in the same column represent significant differences (*p*-value < 0.05).

**Figure 4.** G and G" (**a**) and loss factor (G"/G ) (**b**) assessed as a function of angular frequency in gelatinized starch samples at 70 ◦C.

The effect of CNC on the viscoelasticity of our starch blends is shown in Figure 5, and selected values of G , G" and the loss factor assessed at 10 rad/s and 70 ◦C are presented in Table 3. Our results show that the effect of CNC on the G is different depending on the starch source. Thus, CNC produced a significant increase in G in potato starch and waxy starch (*p*-value < 0.05) but a significant decrease in wheat starch. However, this effect was not proportional to the CNC concentration (Table 3). Similar behavior was observed with G" data. The behavior of G and G" were well reflected by the loss factor data which increased in the case of potato and wheat starch as a function of CNC concentration and decreased in waxy starch in the presence of CNC.

#### *3.3. Thermal Properties of Gelatinized Starch–CNC Blends*

Both the gelatinization temperature (◦C) and gelatinization enthalpy (J/g starch) of starch–CNC blends are shown in Figure 6. The presence of CNC had little effect on the gelatinization temperature recorded from the onset of the endothermic peak associated with starch granule swelling, which agrees with the behavior of pasting temperature assessed by RVA (Table 1). As far as enthalpy is concerned, significant changes were only observed in potato starch, where the presence of CNC produced a decrease in enthalpy but without significant differences (*p*-value > 0.05) among CNC concentrations. In both wheat and waxy starch, the presence of CNC did not change the value of energy necessary to trigger the granule swelling and disruption.

**Figure 5.** G , G" and loss factor (G"/G ) assessed as a function of angular frequency in gelatinized starch samples at 70 ◦C and at different concentrations of CNCs: (**a**) potato; (**b**) wheat; and (**c**) waxy maize. Top plots correspond to a frequency sweep as a function of shear rate, whereas the bottom plots correspond to the loss factor as a function of shear rate.

**Table 3.** G , G" and loss factor of starch–CNC blends assessed at an angular frequency of 10 rad/s and 70 ◦C. Values in brackets correspond to standard deviation. Different upper letters in the same column represent significant differences (*p*-value < 0.05).

**Figure 6.** Gelatinization temperature (**a**) and enthalpy (**b**) of starch from different sources (potato, wheat and waxy) as a function of CNC concentration. Continuous lines only correspond to guide the eye.

#### **4. Discussion**

Differences in the viscosity patterns among starches showed in Figure 1 can be explained by polymorphism expressed by each starch (e.g., A-type in case of wheat and waxy, B-type for potato), which are dependent on the source, the hierarchical structure and lamellar organization in native granules and the branch-chain length of amylopectin [20]. On the other hand, the assessment of pasting parameters (Table 1) can be used as an approach to understand the effect of CNCs on the gelatinization of starch. Thus, the decrease in peak viscosity suggests that the mechanism of granule swelling and disruption is affected by the presence of CNCs, although this behavior would be dependent of the starch source. The literature has only partially reported the effect of CNCs (or other nanowhiskers) on the pasting properties of gelatinized starch, but with contradictory results. For instance, Cui et al. [21] described that CNCs produced a significant increase in peak viscosity in both waxy maize and sweet potato starch, which was attributed to a cross-linking effect via hydrogen bonding between CNCs and starch granules, resulting in a reduced degree of granule swelling. However, Ji et al. [22] found that chitin nanowhiskers significantly reduced the peak viscosity in potato starch, explaining this behavior by an effect of replacement of starch with chitin nanowhiskers causing a dilution of the concentration of starch, and decreasing the peak viscosity. George et al. [23] recently suggested that a lower hydration property restricts the granule swelling and thereby reduces the peak viscosity. Likewise, the literature hypothesized about the ability of cellulose particles to bind water or compete for free water with leached amylose and ungelatinized granules affecting hydration, mobility and granule swelling [19,24]. This could be another reason why in our study the presence of CNCs reduced the peak viscosity.

On the other hand, the pasting temperature was not changed by CNCs, suggesting that the process by which the starch granules begin to swell is not modified by the presence of CNCs, regardless of the starch origin. These results agree with the study of Cui et al. [21] who reported that the presence of CNCs did not modify the pasting temperature in normal maize, waxy maize and sweet potato starches. However, that study was carried out using samples with marked differences in starch concentration, which resulted in values of the pasting temperature which were identical between waxy and potato. Previously, Ji et al. [22] reported that chitin-based nanowhiskers did not change the pasting temperature of maize starch. Most recently, George et al. [23] reported that the addition of starch nanocrystals in concentrations of up to 15% did not produce significant changes in the pasting temperature of rice and wheat starch.

The hold viscosity was shown to be significantly lower at the highest CNC concentration tested in potato and waxy starch, which agree with the data of apparent viscosity (Table 2). The literature lacks works that describe changes in viscosity in gelatinized starch by the effect of nanoparticles and nanocrystals. BeMiller [25] reviewed the effect of different hydrocolloids on the pasting, paste and gel properties of different starch–hydrocolloids composites, finding that changes in viscosity in gelatinized starches would be in dependent of the hydrocolloid molecular size, and concluding a possible direct correlation between the molecular weight and increase in viscosity. Most recently, Ji et al. [22] reported that the presence of chitin nanowhiskers did not modify the hold viscosity in maize and potato starch pastes, which was explained by a possible effect of the interaction between chitin nanowhiskers and starch molecules via hydrogen bonds among the OH groups of nanowhiskers and starches. Likewise, the literature reported that a significant decrease in breakdown viscosity would suggest a reduced capacity for the swelling of starch granules [26]. This could be the case in the data reported in Table 1; however, the evaluation of the relative decrease in viscosity during gelatinization resulted in potato starch showing a decrease from values of 3.80 to 1.26 by the effect of CNCs (Table 1). In the case of wheat and waxy maize starch, the values of relative decrease remained constants except for the highest CNC concentration, which was lower in both cases. These results support the fact that CNCs mainly modify the RVA pattern and pasting properties in potato starch.

Regarding the apparent viscosity behavior of starch at 70 ◦C, our results show a significant higher viscosity in potato starch (Figure 2). This behavior was correlated with the substantially longer branch-chains length of the potato starch which once leached out during gelatinization to offer higher resistance to flow [20]. The non-Newtonian behavior of starchy pastes can be explained by the alignment effect of starchy particles in the direction of the shear during the analysis, as has been reported in the literature [20,27–29]. The increase in shear stress as a function of the shear rate (Figure 2b) associated with changes in viscosity has been related with the solid–liquid character of the gelatinized suspensions [30], as well with the increase in interaction between starch particles and fibrils. Agglomerates or network structures able to be broken or aligned during rheological measurements result in higher shear stress values [24,27].

Changes in pasting properties can be better understood through the viscoelastic characterization of starch pastes. The results shown in Figure 4 suggest that once gelatinized, potato starch shows a less ordered structure (lower G , less elastic) compared to wheat and waxy starch. In wheat and waxy, the starchy paste is more elastic at lower angular frequencies, suggesting the time-dependence condition of the structural organization of gelatinized starch tested at 70 ◦C. This behavior may help to explain the differences previously found in hold viscosity (Table 1) and apparent viscosity (Figures 3 and 4). Since potato starch was shown to be less organized and less elastic once gelatinized, the viscosity in potato starch is higher than that observed in wheat and waxy starch, which is consistent with the higher values of the loss factor observed in the potato starch. These results suggest that the gelatinization mechanism could be different depending on the starch origin or could be influenced by the polymorph organization presented in the native granule. Ai and Jane [20] have reported that the integrity of a swollen granule after gelatinization is essential to understanding the formation of strong gels. The granules of potato starch readily swell and disperse during gelatinization because of the lack of amylose–lipid complex formation which helps to maintain the integrity of swollen starches [20]. Thus, this behavior agrees with results shown in Figure 5 and Table 3, where gelatinized potato starch showed a lower G and higher loss factor values at 70 ◦C. Similar behavior was reported by Cui et al. [21] whom despite testing the viscoelasticity of different gelatinized starch–CNC gels at 4 ◦C, also showed that sweet potato starch has a lower G and higher loss factor values compared to normal maize and waxy maize starch.

Regarding the effect of CNCs on the viscoelasticity of gelatinized starches assessed at 70 ◦C, our results showed to be strongly dependent on the starch origin. Only a few studies reporting viscoelastic data in starch–CNC blends are available in the literature. For instance, Cui et al. [21] suggested the occurrence of interactions between CNCs and amylose, allowed by the high specific surface area and hydroxyl groups of CNCs. These interactions would explain why, in their study, CNCs delayed retrogradation in both sweet potato and normal maize starch. Our results show that waxy starch would retain structural order after gelatinization due to an increased G and decreased loss factor as a function of CNC concentration (Figure 5, Table 3). In turn, in both potato and wheat starch, the presence of CNCs increased the loss factor, suggesting a less organized structure [31]. Interestingly, CNCs decreased the value of G in wheat starch, whereas in potato starch, CNCs increased G . However, the potato starch showed higher values of the loss factor compared with the wheat starch despite the fact that CNCs also lead to an increase in G" in potato starch (Figure 5, Table 3). This behavior could be explained by the higher interaction amylose–CNC promoted by the leaching of amylose during gelatinization, which in the case of potato, is higher than in other starches, as was proposed by Ai and Jane [20]. Thus, the higher amylose–CNC interaction occurring in potato starch could explain the higher values of G and G", which was not observed in wheat and waxy starches. Future studies of this point should include using IR-spectroscopy or RAMAN spectroscopy to characterize the type and intensity of these interactions.

Thermal characterization by DSC showed a good correlation with rheology data. The fact that the gelatinization temperature (Figure 6a) and pasting temperature (Table 1) of each starch did not show significant differences (*p* > 0.05) suggests that temperature related to the starting point of starch gelatinization is not modified by CNCs. Similar behavior was reported by Cui et al. [21] and Ji et al. [22]. Differences among the values of gelatinization temperature and pasting temperature could be explained because gelatinization temperature by DSC is detected from the point at which the double helices of amylose and amylopectin begin to unfold, whereas the pasting temperature from RVA is detected from the point at which the granule starts to swell [32]. On the other hand, differences detected between the gelatinization enthalpies of different starches also reflect differences in how these starches gelatinize. Thus, higher values of enthalpy in potato starch agree with the fast swelling and higher dispersion because of the absence of the amylose–lipid complex which helps to maintain the integrity of swollen starches [20]. However, the effect of CNCs reducing the value of the gelatinization enthalpy, which has been reported by other authors in the literature [21,33], can have more than one explanation. For example, Cui et al. [21] explained the reduction in enthalpy by the addition of CNCs due to an effect of the inhibition of gelatinization and conservation of double helices. Likewise, the ability of cellulose particles to bind water or compete for free water with leached amylose affecting hydration and granule swelling was hypothesized in the literature [19,26]. However, a potential effect of amylose–CNC interaction as has been suggested by our rheological characterization should not be neglected. Nonetheless, this behavior could help explain the lower enthalpy observed in wheat and waxy starch which were not modified by the presence of CNCs (Figure 6b). In this sense, as has been proposed in the previous section, a complementary analysis carried out by IR-spectroscopy and NMR could help understand this phenomenon that has not been extensively studied to date and may open up a new area of research.

#### **5. Conclusions**

Our results suggest that the presence of CNC produces changes in the mechanism of granule swelling and disruption during gelatinization, along with promoting a certain amylose–CNC interaction, although this behavior would be dependent of the starch origin. Thus, in potato starch, there was a marked decrease in peak viscosity and breakdown viscosity over CNC concentration, both values being consistent with the assessment of relative decrease in viscosity. This behavior was not observed in wheat starch and waxy starch. Hold viscosity measured by RVA agreed with apparent viscosity measured by rotative test, whereas both the peak temperature and gelatinization temperature were not changed by the presence of CNCs in all the starches studied. However, viscoelastic characterization at 70 ◦C showed higher values of loss factor in potato starch–CNC, suggesting a less organized structure after gelatinization, which in turn showed good correlation with the increase in G" at different CNC concentration. Our results are presumably influenced by how potato starch gelatinizes and would not be affected by the presence/absence of amylose, as data from wheat and waxy starch suggest. The amylose–CNC interaction in potato starch could explain the higher values of enthalpy detected by calorimetry. However, complementary studies are needed to define the characteristics of these interactions.

These results can be useful in different technological applications based on the use of starch–nanocellulose composites. For instance, for the design of novel composite materials for packaging, bioplastics, bioprinting and food ingredients, to name a few. The potential impact of these biomaterials on sustainable processes opens interesting perspectives in terms of industrial application.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14081560/s1, Figure S1: G (a) and G" (b) assessed as a function of oscillation strain in gelatinized starch samples at 70 ◦C. Figure S2: G and G" assessed as a function of oscillation strain in gelatinized starch samples at 70 ◦C, and at different concentrations of CNC: (a) potato, (b) wheat and (c) waxy maize.

**Author Contributions:** Conceptualization, P.D.-C.; methodology, P.D.-C.; validation, J.M. and P.D.-C.; formal analysis, J.C., K.R., J.M. and P.D.-C.; investigation, J.C., K.R., J.M. and P.D.-C.; resources, P.D.- C.; data curation, J.C., K.R. and J.M.; writing—original draft preparation, P.D.-C.; writing—review and editing, P.D.-C.; visualization, P.D.-C.; supervision, J.M. and P.D.-C.; project administration, P.D.-C.; funding acquisition, P.D-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Fondo Nacional de Desarrollo Científico y Tecnológico FONDECYT grant number 1191375.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Our study do not report supporting results.

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

#### **References**

*Communication*

### **Super-Hydrophobic Magnetic Fly Ash Coated Polydimethylsiloxane (MFA@PDMS) Sponge as an Absorbent for Rapid and Efficient Oil/Water Separation**

**Mengqi Zhao 1,2,\*, Xiaoqing Ma 1, Yuxi Chao 1, Dejun Chen 1 and Yinnian Liao 1,\***

**Abstract:** In this study, magnetic fly ash was prepared with fly ash and nano-magnetic Fe3O4, obtained by co-precipitation. Then, a magnetic fly ash/polydimethylsiloxane (MFA@PDMS) sponge was prepared via simple dip-coating PDMS containing ethanol in magnetic fly ash aqueous suspension and solidifying, whereby Fe3O4 played a vital role in achieving the uniformity of the FA particle coating on the skeletons of the sponge. The presence of the PDMS matrix made the sponge super-hydrophobic with significant lubricating oil absorption capacity; notably, it took only 10 min for the material to adsorb six times its own weight of n-hexane (oil phase). Moreover, the MFA@PDMS sponge demonstrated outstanding recyclability and stability, since no decline in absorption efficiency was observed after more than eight cycles. Furthermore, the stress–strain curves of 20 compression cycles presented good overlap, i.e., the maximum stress was basically unchanged, and the sponge was restored to its original shape, indicating that it had good mechanical properties, elasticity, and fatigue resistance.

**Keywords:** super hydrophobic; magnetic fly ash; polydimethylsiloxane; oil/water separation

#### **1. Introduction**

With global industrialization, oil spills remain a major source of water pollution and are among the most difficult challenges facing the world today. They are not only harmful to the natural ecosystem, but also have long-term adverse effects on human health and the economy [1,2]. However, treatment methods, such as flotation [3], combustion and linseed oil, have the disadvantages of poor selectivity and low efficiency [4].

The contact angle of water droplets on a lotus leaf is about 156◦. As such, water droplets can easily slide off the surface of a leaf, taking surface pollutants with them and thereby achieving a self-cleaning effect. This phenomenon known as the "lotus leaf effect". Inspired by this natural phenomenon, biomimetic superhydrophobic membrane materials based on polymer materials have been developed through polymerization, vapor/liquid phase deposition, membrane transfer, micro-contact printing, electrostatic spinning and other processes. Superhydrophobic materials have a wide range of applications [5–7]. In recent years, the surface structures of lotus leaves have been mimicked, and interfaces similar to those of hydrophobic biomaterials with completely different wettability properties for oil and water have been designed and synthesized on the basis of surface chemical bionics. Such structures can effectively achieve efficient separation of oil and water. Superhydrophobic materials prepared by mimicking the surface characteristics of natural superhydrophobic materials are collectively known as biomimetic superhydrophobic materials. With the aim of adsorbing organic pollutants and purifying oily wastewater, research on such structures is rapidly growing in scope [8–10].

Superhydrophobicity means that water droplets are spherical on the surface with a contact angle greater than 150 degrees. True intrinsic superhydrophobicity does not exist,

**Citation:** Zhao, M.; Ma, X.; Chao, Y.; Chen, D.; Liao, Y. Super-Hydrophobic Magnetic Fly Ash Coated Polydimethylsiloxane (MFA@PDMS) Sponge as an Absorbent for Rapid and Efficient Oil/Water Separation. *Polymers* **2022**, *14*, 3726. https://doi.org/10.3390/ polym14183726

Academic Editors: Valentina Siracusa, Nadia Lotti, Michelina Soccio and Alexey Iordanskii

Received: 3 August 2022 Accepted: 2 September 2022 Published: 7 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

and the maximum water contact angle for flat materials is only 119 degrees. However, metals, ceramics and polymers can be made superhydrophobic by certain treatments, i.e., creating a suitable surface roughness or through the modification of a low surface energy material. For example, metals do not have superhydrophobic properties, but if their surface is roughened by corrosion etching and the surface energy is reduced by fluorination, a contact angle of more than 150 degrees can be obtained, thus turning them into superhydrophobic materials. In contrast, the surface energy of polymers is usually very low, making it easier to convert them into superhydrophobic materials [11].

For example, non-stick pans are made of polytetrafluoroethylene, which is superhydrophobic as long as the surface is rough. The lower the surface energy of the material (i.e., the more energy the molecules on the surface of the material have compared to the molecules inside), the better the hydrophobicity. When a low surface energy material has a microscopic rough structure, an air film will be formed between the water droplets and the material, preventing water from wetting the surface and thus forming a superhydrophobic state [12].

The surface energy of synthetic polydimethylsiloxane (PDMS) is only 22 mN/m. The compound is fluorine-free, environmentally-friendly and cheap, and it can be made to be highly soluble in organic solvents with curing agents [13,14]. PDMS has Si-O-Si as its main chain and has both organic groups and inorganic structures. Its Si-O bond length and bond angle are large, and it is easy to deform. As such, it can grafted/coated in the preparation of low surface energy biomimetic materials with special wettability properties, making it possible to achieve selective adsorption or filtration of oil–water mixtures [15,16]. Wang [17] prepared a super-hydrophobic PDMS@Fe3O4/MS sponge by an impregnation method. The sponge has the advantages of low cost, simple operation and high oil–water separation efficiency. The same author [18] synthesized a hydrophobic PDMS/reduced graphene oxide composite with an adsorption capacity for n-hexane that was 18.5 times its own weight. Zhu [19] connected cement gel particles with PDMS by covalent bonds. This not only changed the morphology of the particles, but also increased their contact angle, providing a research basis for PDMS organic/inorganic hybrid biomimetic materials. Finally, Yu et al. prepared Fe3O4 nanoparticles by the solvothermal method. They then used ammonium persulfate (APS) as an initiator to synthesize magnetic polystyrene oilabsorbing materials with different coating rates by emulsion polymerization of styrene and divinylbenzene (DVB) at different doses on the nanoparticle surfaces [20].

Fly ash is a kind of solid waste, whose main components are silica and alumina. Fly ash itself has adsorption capacity. Compared with other materials, fly ash has a large specific surface area and a porous structure, and thus, has good adsorption characteristics. The use of fly ash as a raw material has obvious advantages in terms of the required source materials.

The innovation of this study is the preparation of a new composite functional polymeric bionanomaterial, MFA@PDMS, for oily wastewater treatment. Based on the concept of "waste to waste", the natural porous structure of solid waste fly ash was used to construct a PDMS porous skeleton to make it rough and improve its oil–water separation efficiency [21,22]. Magnetic Fe3O4 nanoparticles were used to modify the natural porous structure of the fly ash in order to prepare the magnetic porous skeleton. Then, the superhydrophobic bionanomaterials were combined with the magnetic porous skeleton material coating/resin to prepare fluorine-free oil–water separation polymer bionanomaterials.

The preparation process is simple and the resulting magnetic sponges are super hydrophobic and super lipophilic. Additionally, the magnetic properties of the material make it possible to separate oil–water mixtures by an external magnetic field [23]. Magnetic nanomolecular sieve composites have the advantages of high magnetization performance and surface area. In addition, Fe3O4 nanoparticles were shown to improve the mechanical properties of the sponge, which remained superhydrophobic when it was under tension or compression. The tested sponge also showed good mechanical stability, oil stability and reusability in terms of its superhydrophobicity and oil absorption [21].

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

#### *2.1. Materials*

The fly ash (FA) used in this study was obtained from the Kanas Power Plant in Xinjiang Autonomous Region, China. Its chemical composition was as follows: SiO2 (45.9 wt%), Al2O3 (19.03 wt%), CaO (16.39 wt%), SO3 (6.01 wt%), Fe2O3 (5.65 wt%), K2O (2.07 wt%), MgO (2.02 wt%) and Na2O (0.954 wt%). The PDMS prepolymer and its curing agent were obtained from American Dow Corning. FeCl2·4H2O, FeCl3·6H2O and sodium citrate all came from Macklin Biochemical (Shanghai, China) Co., Ltd. Ammonia water was obtained from Sigma-Aldrich (Shanghai, China) Trading Co., Ltd.

#### *2.2. Preparation of Magnetic Fly Ash/Polydimethylsiloxane (MFA@PDMS) Sponge*

(1) Preparation of magnetic fly ash: First, we poured FeCl2·4H2O (1.72 g) and FeCl3·6H2O (4.72 g) into a three-port flask, added 80 mL distilled water, and stirred for 10 min at 80 ◦C. Then, we added ammonia water dropwise (25 %) until a pH of 10 was reached. Black material could be seen in the process of dropping, and the reaction continued for 30 min after the solution had turned black. Next, 20 mL 0.3 m sodium citrate solution was added and the mixture heated to 90 ◦C for 30 min. We then added about 8 g of fly ash and continued stirring for 2 h. At the end of the reaction, we applied solid–liquid separation with an external magnetic field and repeated washing with distilled water. Then, we dried the compound at 80 ◦C for 12 h and, finally, used mechanical grinding to obtain the magnetic fly ash nanomaterials (FA@Fe3O4).

(2) Preparation of magnetic fly ash @PDMS bionic material: First, we placed 0.20 g magnetic fly ash, 4.00 g PDMS prepolymer and 0.40 g curing agent (10:1) into a beaker. The mixture was stirred, and 2.00 g anhydrous ethanol was added and mixed evenly. We then slowly added 4 mL water, stirred and allowed the mixture to emulsify for 15 min. It was poured into a sand core funnel mold for forming, and then placed in a drying oven at 120 ◦C for curing for 1.5 h to obtain dark brown spongy magnetic material (MFA@PDMS).

#### *2.3. Adsorption Capacity, Separation Efficiency and Reusability Test*

(1) To evaluate the adsorption capacity, a certain mass of the material was first immersed in an organic solvent and weighed after reaching mass absorption equilibrium. The measurement was repeated three times and the average value was taken. The adsorption capacity *Q* of the material was calculated according to Formula (1).

$$Q = \frac{(M\_1 - M\_0)}{M\_0} \tag{1}$$

where *Q* (g/g) is the mass-based adsorption capacity, *M*1 (g) is the mass of the sponge after oil absorption and *M*0 (g) is the mass of the sponge before oil absorption.

(2) Next, we built an oil–water separation device and calculated the oil–water separation efficiency, *R*, of the sponge according to Formula (2).

$$R = \frac{V\_{\text{C}}}{V\_0} \times 100\% \tag{2}$$

where *R* (%) is the oil–water separation efficiency, *Vc* (mL) is the volume of the original solvent or oil collected after separation and *V*0 (mL) is the volume of the original solvent or oil before separation.

(3) The repeatability of the material was initially evaluated using a simple electronic universal testing machine. First, the material was immersed in an organic solvent to achieve maximum absorption and its mass was weighed and recorded as *M*2. Afterwards, the absorbed oil was recovered by mechanical compression and the mass of the material was determined again and recorded as *M*3. The reusability of the sponge was assessed by absorption-compression cycles, each time weighing the material to calculate the adsorption multiplier (i.e., the weight of the material after the adsorption of an organic solvent/the weight of the material itself).

#### *2.4. Wenzel Model*

In Equation (3), *r* represents the roughness factor of the solid surface (*r* > 1), which is actually the magnitude of the ratio of the actual area on the surface to the projected area; *θW* represents the apparent contact angle with the rough surface; and *θY* is the contact angle in an ideal state [12].

$$\frac{\cos\theta\_W = r(\gamma\_{SV} - \gamma\_{SL})}{\gamma\_{LV}} = r \cdot \cos\theta\_Y \tag{3}$$

Under the condition that the solid surface itself is hydrophilic, if the surface roughness increases, the hydrophobicity of the surface will not increase. In contrast, under the condition that the solid surface itself has a certain degree of hydrophobicity, if the surface roughness increases, it will also have a certain effect on the hydrophobicity of the solid surface, and the level of hydrophobicity will naturally increase.

#### *2.5. Determination and Characterization*

The composite samples were analyzed using an X-ray fluorescence spectrometer (XRF, ARL PERFORM'X, Thermo Fisher Scientific, Waltham, MA, USA) and Fourier transformation infrared spectrometer (FTIR, VERTEX 70 RAMI, Bruker Corporation, Billerica, MA, USA). The morphologies of samples were comparatively observed by scanning electronic microscopy (SEM, SU8010, Hitachi, Ltd., Tokyo, Japan). The morphologies of the samples were observed using a transmission electron microscope (TEM, JEM-2100, JEOL Ltd. Akishima City, Tokyo, Japan). Magnetic analyses of the samples were conducted using a vibrating sample magnetometer (VSM, Lake Shore Corporation, Columbus, OH, USA). The stress–strain analyses of the samples were conducted using an electronic universal testing machine (CMT6103, MTS Systems Corporation, Eden Prairie, MN, USA).

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

#### *3.1. Materials Characterization*

#### 3.1.1. TEM Analysis of Nanometer Fe3O4

The sample powder was dried using a 200 nm copper mesh. The electron acceleration voltage of the transmission electron microscope (TEM) was 100 kV.

A TEM image of the magnetic nano-Fe3O4 is shown in Figure 1. As shown, the average particle size of the magnetic nanospheres was 30 nm, and the particle size was uniform.

#### 3.1.2. SEM Analysis

As Figure 2a,b shows, the surface of PDMS was extremely uniform and the folds were extremely smooth, which is the reason why the sponge material had such good mechanical properties and elasticity. Additionally, its internal porous structure increased its oil absorption performance to a certain extent.

**Figure 2.** SEM of (**a**,**b**) PDMS and (**c**,**d**) MFA@PDMS.

As Figure 2c,d shows, the morphology changed significantly with modification with MFA; the smooth surface of the PDMS became rough, which was favorable for hydrophobicity. Additionally, because of the natural porous structure of FA, the MFA@PDMS had a rough, honeycomb porous structure, which undoubtedly increased the hydrophobicity of the sponge surface and the oil absorption ability of the sponge.

#### 3.1.3. FT-IR Analysis

The Fourier transform infrared spectrum (FT-IR) of the MFA@PDMS is shown in Figure 3. The absorption peak at 2964 cm−1 corresponds to -CH3 stretching vibrations; that at 1262 cm−1 was attributed to the sharp symmetric deformation vibration peak of Si-CH3; that at 793 cm−1 corresponded to that Si-C tensile vibration peak [23]; that near 1083 cm−1 was attributed to the asymmetric stretching vibrations of Si-O-Si bond and Si-O-Al bonds, which was caused by the polymerization of the fly ash aluminosilicate or silicon-oxygen tetrahedron structure. Finally, a crystal lattice absorption peak of Fe3O4 appeared at 599 cm−1, indicating that the MFA@PDMS material had been prepared successfully.

**Figure 3.** FT-IR Diagram of MFA@PDMS.

3.1.4. Characterization of Magnetic Properties on MFA@PDMS Sponge

The vibrating sample magnetometer (VSM) test results for nano-Fe3O4 and MFA@PDMS sponge are shown in Figure 4. As shown, their coercivity was low, and they demonstrated super-paramagnetism. Figure 5 is a graph comparing the natural sedimentation separation and magnetic field separation of the FA@Fe3O4 suspension.

**Figure 4.** Hysteresis loop of Fe3O4 and MFA@PDMS.

**Figure 5.** Solid–liquid separation process of FA@Fe3O4. (**a**) Suspension; (**b**) Natural sedimentation separation; (**c**) Magnetic field separation.

#### *3.2. Hydrophobicity of MFA@PDMS Sponge*

Deionized water dyed with blue ink (aqueous phase) and hexane dyed with oil-soluble black (oil phase) were dropped on the surface of the MFA@PDMS sponge (the volume ratio of hexane to water was 1:1). The experimental results are shown in Figure 6. The deionized water with blue ink formed a typical spherical shape, revealing the superhydrophobicity of the surface of the material. The surface contact angle was calculated to be greater than 150◦, while the black hexane penetrated the interior of the parent body. A material with hydrophobic characteristics cannot be wetted by water, but can be wetted by oil. It can be seen that the sponge material had good hydrophobicity to aqueous solution, reflecting its good waterproof performance; this is also key its selectivity for oil–water separation.

**Figure 6.** Hydrophobic Properties of MFA@PDMS sponge.

*3.3. Oil–Water Separation Performance of MFA@PDMS Sponge* 3.3.1. Separation Performance for Immiscible Oil/Water

In order to verify the oil–water separation capability of the MFA@PDMS sponge, an experiment was designed (see Figure 7). The MFA@PDMS sponge was placed in a normal hexane aqueous solution containing Sudan red II dye. The hexane solution was then moved to an oily area using a magnet. It was observed that the red normal hexane was quickly absorbed by the sponge (the red circles correspond to the locations of the red hexane), with the oil-bearing water quickly becoming transparent, indicating that that material has excellent oil–water separation capability (the separation efficiency can reach more than 95%), can effectively and quickly remove n-hexane (oil phase) from water, and can be remotely controlled via an external magnetic field, thereby facilitating the separation, recovery and transportation of oils.

**Figure 7.** Oil–water separation using the MFA @ PDMS sponge.

According to the experimental process shown in Figure 8, the adsorption times of pure PDMS, FA@PDMS, Fe3O4@PDMS and MFA@PDMS sponges for n-hexane were tested; the results are shown in Figure 8. As indicated, the adsorption multiple of the MFA@PDMS sponge was the highest, reaching six times of its own weight, indicating that the addition of MFA significantly improved the oil–water separation effect of the PDMS.

**Figure 8.** Comparison of adsorption times.

3.3.2. Separation Performance for Oil–Water Emulsion

In general, the particle size of the dispersed phase of oil–water emulsions is much smaller than 20 μm, and a stable protective layer is formed on the surface of droplet molecules. As such, it is difficult to break emulsions, making oil–water separations extremely difficult. In this study, the separation efficiency of MFA@PDMS for different types of emulsified oils (n-hexane, phenol and canola oil) was investigated via the experimental method shown in Figure 9. The results are shown in Figure 10.

**Figure 9.** Schematic diagram of oil–water separation process of emulsion mixture.

**Figure 10.** Schematic diagram of emulsion oil–water separation efficiency changes over time.

This oil–water separation device was based on the hydrophobicity and lipophilicity of the MFA@PDMS based material. The MFA@PDMS was clamped in the middle of the left tube and the oil–water mixture was poured into the wide-mouth collection container on the right side. The red oil quickly passed through the MFA@PDMS and flowed into the collection container on the lower left side, while the blue deionized water was blocked on the top side of the MFA@PDMS. Finally, the oil–water mixture was successfully separated. When the oil–water mixture came into contact with the MFA@PDMS surface, its superhydrophobic and super lipophilic properties caused the oil contaminants to penetrate and pass through the material instantly, while the aqueous solution was blocked by its hydrophobic nature. In this study, after each separation, the sample was washed and dried before being reused for the next oil–water separation.

As shown in Figure 10, the separation efficiency of magnetic MFA@PDMS sponge for n-hexane emulsified oil was the highest, reaching 97.12%, followed by phenol, which reached 95.27%. This was due to the unique structure of fly ash, i.e., its large specific surface area, porosity and abundant aluminum-silicon components, as well as the superhydrophobic performance of PDMS, which can attract organic molecules and chain-like structures to the inner surface of sponge materials through π-π dispersion interactions and the donor receptor effect [6].

#### *3.4. Reuse of MFA@PDMS Sponge*

The experiment shown in Figure 11 was designed to characterize the reutilization performance of the sponge bionic material. The sponge material was put into n-hexane dyed with Sudan red II, and it was observed that its thickness increased from 0.6 cm to 1.2 cm after only 10 min. We then removed the sponge bionic material and weighed it to calculate its adsorption multiple (i.e., the weight of the material after absorbing the normal hexane/the weight of the material). The oil phase could be extruded by simple compression, and the material could be reused within 30 min after extrusion. Over eight adsorption-extrusion-drying tests, the oil absorption capacity did not decrease significantly, showing good reusability.

**Figure 11.** Flexible deformation of materials during oil absorption-extrusion-drying process on MFA@PDMS sponge.

An H5KT-type static mechanical tester was used for our stress–strain performance analysis. The compressive stress–strain relationships under single and 20 cyclic quasi-static compressive loadings are shown in Figures 12 and 13, respectively (the compression rate was 50 mm/min).

As shown in Figures 12 and 13, the maximum stress of the material was 446.9 kPa before adsorption on the sponge and 410 kPa afterwards, indicating that the material has good recyclability. The stress–strain curves of 20 compression cycles showed good overlap, and the maximum stress was basically unchanged, indicating that the sponge has good mechanical properties, elasticity, and fatigue resistance.

**Figure 12.** Stress–strain diagram of MFA@PDMS sponge material during a single cycle.

**Figure 13.** Stress–strain diagram of MFA@PDMS sponge over 20 cycles.

#### **4. Conclusions**

In this study, magnetic FA was used to construct a flexible PDMS porous structure, and a MFA@PDMS sponge for oil–water separation was successfully prepared. The sponge was shown to be able to adsorb six times its own weight of n-hexane in only 10 min. It could remotely process oil-bearing waters under the action of an external magnetic field, making it an effective, fast and convenient approach. The MFA@PDMS can be reused by applying a simple oil absorption-extrusion-drying operation which takes around 30 min, after which the oil–water separation performance is not significantly reduced. The stress–strain curves of 20 compression cycles for the sponge showed good overlap, and the maximum stress was basically unchanged, i.e., the sponge was restored to its original shape, indicating that it has good mechanical properties, elasticity, and fatigue resistance.

**Author Contributions:** Conceptualization and methodology, M.Z. and Y.L.; methodology, M.Z.; formal analysis, M.Z. and X.M.; investigation, M.Z. and X.M.; resources, Y.C. and D.C.; data curation, M.Z. and Y.L.; writing—original draft preparation, M.Z.; writing—review and editing, M.Z. and X.M.; visualization, X.M. and D.C.; supervision, Y.C. and D.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research were funded by the National Natural Science Foundation of China (21968035, U2003123) and the Provincial Natural Science Foundation of Xinjiang Uygur Autonomous Region (2020D01C021).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Meiling Song from Shiyanjia Lab (www. shiyanjia.com (accessed on 23 Mar 2022)) for the VSM analysis.

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

#### **References**

MDPI St. Alban-Anlage 66 4052 Basel Switzerland www.mdpi.com

MDPI Books Editorial Office E-mail<EMAIL_ADDRESS>www.mdpi.com/books

Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Academic Open Access Publishing

mdpi.com ISBN 978-3-0365-8579-6