Abstract

Synthesis of cerium oxide (CeO2) nanoparticles (NPs) via biological approach has received a lot of interest to reduce the harmful effects of chemical synthesis. In the present study, Colocasia esculenta leaf extract facilitated the preparation of CeO2-NPs by using the sol-gel technique. The crystal structural of CeO2-NPs was proven by X-ray powder diffraction (XRD) investigation to be cubic with size of 2.94 nm according to Debye–Scherrer equation. As demonstrated in the transmission electron microscopy (TEM) image, CeO2-NPs have a spherical form with an average size of 2.04 nm which is almost consistent with a finding from XRD analysis. Energy dispersive X-ray (EDX) measurements exhibited high-intensity peaks attributed to Ce and oxygen and further proved the creation of CeO2-NPs. The Fourier transform infrared spectroscopy (FTIR) analysis revealed the presence of Ce-O stretching, indicating the formation of CeO2-NPs. Functional groups of O-H, C-O, and C=O peaks were found in a spectrum due to the phytochemical components that were responsible for reducing and stabilizing during the synthesis process of CeO2-NPs. The examined UV-visible spectra exhibited the absorbance peak at 213 nm. The synthesized NPs produced in this study were further investigated for mung bean seed germination, whereby the influence of grain germination and growth rate demonstrated the significant finding.

1. Introduction

Nanoparticles (NPs) are defined as an atom having a diameter range of 1 nm–100 nm [1]. A variety of NPs were fabricated in the past few years such as titanium oxide NPs [2], silver NPs [3], silica NPs [4], iron oxide NPs [5], and copper NPs [6]. Among them, cerium oxide NPs (CeO2-NPs) owing to their excellent electronic, eco-friendly properties, and tremendously growing catalytic applications, have exhibited tremendous contribution diversity of applications such as therapeutic agent [7], antibacterial agent [8], for impaired diabetic wound healing [9], agent for anticancer [10], and fuel cell [11]. Among these fields, attention should be given to an agriculture field as they play an important role for fiscal economy to many countries, however suffer from the insufficient production of market supply to meet a growing demand of crops plants worldwide. As a solution, fertilizers have been introduced to crop plants to boost the crop production. However, the usage of excess fertilizer to plants creates a future problem to the ecology and environment. Therefore, scientists recommended reducing the quantity of fertilizer to plant and soil to secure sustainability of nature. The efforts are made possible by using nanofertilizers such as zinc oxide NPs [12, 13], copper NPs [14], and iron oxide NPs [15]. To date, NPs display a promising platform to boost plant growth of cabbage [16], maize [17], mung bean [18], and rice [19]. Despite the advantages of the nanosize of cerium oxide, their investigation as nanofertilizer is still limited.

The common approach to synthesis CeO2-NPs are through physical and chemical approaches. However, it has been observed that the aforementioned approaches for the synthesis of CeO2-NPs had several limitations, including the use of toxic chemicals and high-tech automation and incur high cost of chemicals. Of these, biological approach offers a “green” synthesis method with inexpensive method by using abundant natural biological species such as fungus [17], bacteria [20], plants [14], and algae [21]. They act as a reducing agent, stabilizer, and capping agent in the synthesis route which have the potential to substitute expensive chemical capping agents. Previous study had utilized triethanolamine (TEA), ethylene diamine tetra acetic acid (EDTA), tetraethylammonium bromide (TEABr) [22], ethylenediamine (EDA), hexamethylediamine (HTMA) [23], 1-dodecyl-3-methylimidazolium bromide [24], tri-n-octyl phosphine oxide (TOPO), and dodecyle amine (DDA) [25] due to their available bulky and steric hindrance structures. The traditional sol-gel procedures are commonly used to synthesis NPs, but they have various drawbacks, including self-aggregation, uneven distribution of particle size and shape, and nanocluster formation. By using an appropriate capping agent throughout the synthesis phases, agglomeration effects and other limitations of the sol-gel method can be easily overcome [26].

The “green” synthesis of NPs had gain attention in the past two decades to secure environment and achieve sustainability. The leaves waste from Colocasia esculenta (C. esculenta), commonly known as Taro, generate a biowaste from leaves which are plentifully obtainable in Malaysia without any profitable application. As compared to other root crops, C. esculenta produces highly nutritious leaves (petiole and blades), which are high in carbohydrates, proteins, minerals, vitamins, and other phytonutrients with antioxidant potential [27]. These advantages allow them to act as reducing and stabilizing agents in the synthesis of CeO2-NPs. The plant has been reported as reducing and stabilizing agents in the earlier work by Inamuddin and Kanchi [28]. In their work, C. esculenta had successfully impacted on the synthesis of silver NPs indicating the great potential for biological approach in NPs synthesis [28]. To the best of author’s knowledge, no research had been published previously on the potential of their leaves extract to stabilize the “green” synthesis of CeO2-NPs. Regardless of the fact that the plants are rich in nutrient [29], one of the earliest domesticated plants [27] and substantially adaptable, Taro is still regarded as an orphan crop. Therefore, the present study will synthesize CeO2-NPs by using leaves extract from C. esculenta for mung bean (Vigna radiata) seed germination. The morphology, structural, size, and optical properties of synthesized CeO2-NPs were investigated in detail.

2. Experimental

2.1. Chemicals and Reagents

Cerium (III) nitrate hexahydrate [Ce(NO3)3 · 6H2O] was purchased from Sigma-Aldrich Corporation (United States). Analytical grade of ethanol was from Chemiz (M) Sdn. Bhd. (Malaysia). The chemicals were used without further purifications. The deionized water was used throughout the experiment during synthesis and germinations.

2.2. Preparation of C. esculenta Leaf Extract

Leaves from C. esculenta were collected from Malaysian Agricultural Research and Development Institute (MARDI), Selangor, and thoroughly washed, dried under shed, and finely ground to obtain homogeneous and fine powder. The powder was kept sealed in a plastic petri dish until further use. Then, 10 g powdered leaves were heated in 100 mL of deionized water for 15 min at 80°C and then incubated at room temperature overnight. The resulting solution was filtered by using a cotton cloth to remove any residue. The filtrate was used for the synthesis of CeO2-NPs.

2.3. Synthesis of CeO2-NPs

The synthesis of CeO2-NPs followed a method by Mathew et al. [18] with a slight modification. C. esculenta leaf extract of 100 mL was mixed with 0.11 M of Ce(NO3)3 · 6H2O to prepare a solution. The solution was constantly stirred with a magnetic stirrer at 80°C for 4 h. The resulting solution was centrifuged. Water and ethanol were added few times during centrifuge. The collected CeO2-NPs was poured onto a glass plate, oven dried at 60°C overnight, and calcined at 400°C for 3 h. The obtained yellow powder NPs were ground and kept in a seal bottle prior to characterization. The schematic representation of CeO2-NPs synthesis through C. esculenta leaf extract is demonstrated in Figure 1.

2.4. Characterizations of CeO2-NPs

The synthesized NPs were characterized by using T80+ UV/Vis spectrophotometer (PG Instruments) between 200 nm and 600 nm. In X-rays, powder diffraction (PAN analytical X-ray diffractometer operated at 45 kV and Rayon X on 40 mA (Cu-Kα radiation 1.5 Å)) in the 2θ range from 20° to 80° was used to evaluate their crystalline nature and particle sizes. Field emission scanning electron microscopy (FESEM) with EDX-analysis (MERLIN Compact) and transmission electron microscopes (Talos L120C) were performed for surface morphology and shape. The structural changes of CeO2-NPs were determined by using Fourier transform infrared spectroscopy (FTIR) (Perkin Elmer) in the 4000 cm−1–400 cm−1 range.

2.5. Germination Assay

Seed germination assay followed a method by Mathew et al. [18] with a slight modification. In brief, mung bean (Vigna radiata) seeds were placed in a beaker containing distilled water for 2 min-3 min for cleansing purpose and left to dry on papers for 10 min at room temperature. Later, 15 seeds of similar size were selected and immersed in amber bottles that contained different concentrations of CeO2-NPs for 2 h. A control was made by placing 15 seeds inside the amber bottle that contained only distilled water. The serial dilution of required concentration was prepared according to Awan et al. [30]. The stock concentrations of CeO2-NPs were made by using 50 mg in 1 mL of deionized water. Then, a dilution to 0.5 mg·mL−1, 1.0 mg·mL−1, 1.5 mg·mL−1, 2.0 mg·mL−1, and 2.5 mg·mL−1 was conducted by using 10 mL of deionized water to immerse seeds of Vigna radiata. The seeds were spread on moistened cotton surfaces at a rate of 15 seeds per plate. Seed germination was observed daily for 7 days after the germination test was started. Data obtained from the inhibition zone were analysed by using analysis of variance (ANOVA). Tukey’s test was performed to compare the mean differences among treatment at probability level of 0.05 by using SPSS Statistics version 23.

3. Results and Discussions

3.1. Synthesis and Characterization of CeO2-NPs

The formation of CeO2-NPs was indicated by the formation of a thicker solution after 0.11 M of Ce(NO3)3 · 6H2O solution was added to 100 mL of C. esculenta leaf extract, stirred for 4 h at 80°C, and revealed the gradual reduction of Ce4+ to Ce0. According to Utara et al. [31] and Calvache-Muñoz et al. [32], the mechanism for the formation of CeO2-NPs is shown as follows:

When dissolved in plant extract, the precursor dissociated into Ce3+, 3NO3, and 6H2O. Further reaction would produce Ce(OH)3+ as in Step 2. In Step 3, Ce that had oxidation number of +4 from Ce(OH)3+ reacted with hydroxide and water to form Ce(OH)4⋅xH2O. Heating process at Step 4 would produce Ce(OH)4 as the process would remove water from the molecule. Calcination was carried out to completely remove hydroxide from Ce(OH)4 to produce CeO2-NPs.

3.2. UV-Vis Spectroscopy

UV-vis spectroscopy was utilized to examine the formation of CeO2-NPs from the plant extract at 200 nm–600 nm wavelength as displayed in Figure 2. The spectrum exhibited an absorbance peak at 213 nm indicating the development of CeO2-NPs. Based on a previous study, common absorbance peak of CeO2-NPs was found at 332 nm when citrate-nitrate sol-gel method was utilised [33]. Another possible absorption peak was found at 284 nm when CeO2-NPs were synthesized by using hexamethylenetetramine as demonstrated by Parimi et al. [34]. Likewise, Arumugam et al. synthesized CeO2-NPs using Gloriosa superba L. leaf extract, and the NPs exhibited the absorption peak at 297 nm [35]. In the present study, the absorption band occurs at a lower wavelength known as blue shift phenomenon. This was due to the small size of CeO2-NPs that causes this effect as experienced by Krishnamoorthy et al. [36]. In addition to the absorbance, the formation of CeO2-NPs was confirmed visually through the yellowish powder of final NPs after furnace. Eka Putri et al. [37] synthesized CeO2-NPs by using Moringa oleifera leaf extract as a natural bio-reducing agent to reduce a precursor to produce CeO2-NPs. According to their study, the color change was a visible production indicator of CeO2-NPs. As a result, the role of leaf extract as a reducing agent had been confirmed. Another function of Taro’s plant extract serves as a capping agent, whereby it also assists to keep nanoparticles growing and manage their size; hence, it serves as a stabilizer [37].

3.3. XRD Analysis

The X-ray diffraction technique was used to determine the crystalline structure and size of CeO2-NPs. Figure 3 shows the finer and more persistent diffraction peaks demonstrated the crystallinity of CeO2-NPs. CeO2-NPs did not contain any contaminants, as evidenced by the absence of any other diffraction peaks from other species. The X-ray diffraction spectra presented characteristic diffraction peaks at 11.77°C, 28.89°C, 33.68°C, 48.14°C, 57.16°C, 71.07°C, 77.27°C, and 88.37°C. The crystal size of CeO2-NPs, calculated by using Debye–Scherrer equation, was found to be 2.94 nm.where k is the Scherrer constant having a value 0.94, λ is the wavelength of X- ray, β is half-width at half- maximum (FWHM) data with intensity and position is provided by XRD patterns, θ is the Bragg diffraction angle, and D is the particle size [38]. The XRD diffraction patterns of CeO2-NPs were found in accordance with the finding from Ahmad et al. [39], whereby Elaeis guineensis leaves were used as a reducing and stabilizing agent in the formation of CeO2-NPs [39]. In addition, another study by Iqbal et al. [40] utilized Phoenix dactylifera plant extract demonstrated similar XRD diffraction pattern of CeO2-NPs [40].

3.4. FESEM-EDX Analysis

FESEM analysis evaluated morphological image of CeO2-NPs synthesized in leaf extract, crystallography, and evaluation of gradient chemical composition of the NPs [41]. Figure 4 shows a FESEM image of CeO2-NPs produced from C. esculenta leaf extract. As demonstrated in Figures 4(a) and 4(b), the produced NPs were well segregated and very porous. The present NPs exhibited irregular and angular nanostructure with high porous network, whereby proved that the aggregation of NPs was reduced by C. esculenta leaf extract. Almost a similar finding was reported by the authors of [42] that utilized an orange peel extract to use as a capping as well as reducing agent for synthesis of titanium oxide NPs. The EDX spectrum confirmed the chemical compositions and purity of CeO2-NPs produced (Figure 4(c)). The strong cerium (Ce) peaks between 1.0 and 6 keV arise, according to the research. The presence of cerium and oxygen in synthesized NPs are shown by the spectrum peak. This was in accordance to the finding by Mary Isabella Sonali et al. [33] who synthesized CeO2-NPs by using a bacterial strain. Another possible peak was contributed by carbon (C), potassium (K), phosphorus (P), sulfur (S), calcium (Ca), and chlorine (Cl) at a very low percentage (2% and below). The same finding was observed in the work reported by [39], in the synthesis of CeO2-NPs by using Elaeis guineensis (oil palm) leaves, whereby the author reported the presence of C, P, and K in their NPs.

3.5. TEM Analysis

The morphological properties and average grain size of CeO2-NPs made from C. esculenta leaf extract were examined by using transmission electron microscopy. TEM micrographs of produced CeO2-NPs at scales of 10 nm shown in Figure 5(a). The TEM images revealed that the sample was completely invented of spherical, smooth, and far less agglomerated particles. Figure 5(b) shown a histogram obtained for the distribution of CeO2-NPs at 10 nm size by using 50 particles. The average particle size was found to be 2.04 nm, which was found to be consistent with Scherrer’s formula, based on XRD pattern. The tiny size was likely owing to the superior capping agent found in C. esculenta leaf extract, which prevented overagglomeration during the nucleation and ageing stages of CeO2-NP production. Koyyati et al. [43] reported a similar finding in the creation of zinc oxide NPs by using radish (Raphanus sativus) leaves, whereby very small NPs of 2 nm were formed, indicating the plant extract are rich in phytochemical elements to operate as a capping agent to control the size of NPs.

3.6. FTIR Analysis

FTIR spectra were obtained to identify the functional groups of CeO2-NPs produced from C. esculenta leaf extract (Figure 6). Results of FTIR study in leaves extract exhibited transmittance peaks located at about 3431 cm−1, indicating the presence of O-H functional group. The significant functional group was observed at 1634 cm−1 (C=O) due to the presence of carboxylic acids and esters from phytochemical constituent from the plant extract. The presence of peaks at around 3400 cm−1 and 1600 cm−1 were well agreed by the authors of [40] who reported the synthesis of CeO2-NPs in five different extracts of Coriandrum sativum (Coriander), Phoenix dactylifera (Ajwa dates), Camellia sinensis (green tea), Ocimum tenuiflorum (Black tulsi), and Hibiscus flower. In addition, spectrum of CeO2-NPs revealed the peaks observed at 448 cm−1 confirmed the formation of CeO2-NPs caused by the bond of Ce-O. This finding was in agreement to (Vasudevan) [44], whereby CeO2-NPs were successfully synthesized by using Justicia Adathoda leaves extract, indicated by the presence of peak at 500 cm−1. Likely, similar observation was reported by Parvathy et al. [45] who used Artabotrys hexapetalus leaf extract to prepare CeO2-NPs. Significant peaks at 3405 cm−1, 1634 cm−1, and 1077 cm−1 were found similar with the work reported by Butt et al. [46] by using petals of Cinnamon-like bark Cassia glauca as a capping agent in the synthesis of CeO2-NPs.

3.7. Effect on CeO2-NPs Suspensions on Seedling Growth

In recent years, nanofertilizer of CeO2-NPs had been reported to enhance the growth of plants. For example, Abdulhameed et al. [16] produced CeO2-NPs by using laser ablation which yielded a very significant contribution to the growth of cabbage. Similarly, Mary Isabella Sonali et al. studied CeO2-NPs of a germination of fenugreek seeds [33], whereby only 75 ppm of nanosized CeO2-NPs in nanocomposite, the NPs were able to increase the shoot and root length of fenugreek plant. The present works were subsequently found to be in accordance to the previous findings as presented in Figure 7 and Table 1. Results showed that there was a significant difference () between concentration of NPs with root and shoot length of mung bean seedling growth. The increasing concentration of CeO2-NPs resulted in a notable increased length of root and shoot growth. Seed treated with CeO2-NPs was found with increased value of root and shoot length as compared to control (untreated seeds). After 7 days of seedling growth, the optimum growth response for root and shoot in mung bean seedling was recorded at 1 mg/mL concentration with 2.81 ± 1.81 and 16.5 ± 1.41, respectively. This similar phenomenon also was observed in a study by Jayarambabu et al. [47] by using a similar plant seed, however with different concentrations and types of NPs. However, it was found that greater CeO2-NP concentrations resulted in a considerable reduction in root and shoot length. The hazardous amount of NPs could explain the decline in root and shoot growth at higher doses. This was strong evidence that mung bean seedlings responded to NPs in a limited range, above which hazardous levels were obtained, resulting in growth reduction. Results were aligned with the previous study reported by López-Moreno et al. [48, 49] which showed an increasing root and shoot length in Zea mays and Glycine mays L. seedling treated by CeO2-NP, respectively.

4. Conclusion

In this work, CeO2-NPs were successfully prepared by using plant waste from Colocasia esculenta. Based on the characterization via XRD, the crystal size of CeO2-NPs, calculated by using Debye–Scherrer equation, was found to be 2.94 nm and was found consistent with analysis from TEM. The CeO2-NPs were found promising in mung bean (Vigna radiata) seed germination. In future, cytotoxicity study will be examined after the germination to study the toxic effects towards an optimized concentration.

Data Availability

The experimental data used to support the findings of the study are included in the paper.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors would like to thank Universiti Teknologi MARA Shah Alam and Universiti Teknologi MARA Negeri Sembilan Branch for the assistance and facilities provided throughout the research. The authors acknowledge the Centre for Research and Instrumentation Management (CRIM) for the analytical services given. The chemicals and facilities were provided by Universiti Teknologi MARA Shah Alam and Universiti Teknologi MARA Negeri Sembilan Branch.