Green synthesis and characterization of Fe3O4 nanoparticles using Chlorella-K01 extract for potential enhancement of plant growth stimulating and antifungal activity

Iron oxide nanoparticles were successfully prepared using a green approach with microalgal extract in an alkaline medium. Fe₃O₄-NPs was created using a microalgal extract in a quick, cost-effective, and environmentally safe way¹⁹. According to El-Kassas et al. (2017), proteins and polyphenols from Chlorella K01 catalyse the reduction of iron ions into nanoparticles, and polysaccharides stabilize Fe₃O₄-NPs²⁷. Chlorella K01 extract contains 632 ± 195 mg ml⁻¹ of protein, 39.59 ± 3.04 mg ml⁻¹ of carbohydrate and 0.12 ± 0.007% of polyphenol content. Therefore, the extract can be used for the bio fabrication of Fe₃O₄-NPs. 1 g of dry algal powder yielded 16 mg at pH-6, 645 mg at pH-8, 703 mg at pH-10 and 829 mg at pH-12 of Fe₃O₄-NPs. Figure 1 illustrates one month old Chlorella K01 culture, aqueous Fe₃O₄-NPs solution and Crude Fe₃O₄-NPs.

One month old Chlorella K01 culture (A) and synthesized Fe₃O₄-NPs solution (B), Crude Fe₃O₄-NPs (C).

According to the results of the in vitro germination test, Fe₃O₄-NPs synthesized at pH-12 showed significantly higher germination rates (P ≤ 0.05) and was therefore used for further investigation (Fig. 2). The description of the crops used in this experiment, as well as their germination activity, is shown in Fig. 3. In terms of the effect of different Fe₃O₄-NPs concentrations on seed germination, Fe₃O₄-NP-treated seeds (1 mg ml⁻¹) had a higher germination rate, a higher vigor index, and a notable increase in seedling shoot and root formations (P ≤ 0.05) among the crops than GA treated seeds and control seed (Figs. 4, 5). When compared to the positive and negative control seedlings, the Fe₃O₄-NPs treatments had a higher germination rate, root length, and vigor index (P ≤ 0.05). Among the crops tested, green gram demonstrated the most remarkable plant growth and vigor index (Figs. 3, 5). Fe nanoparticles have been shown to have a negative effect on the germination process and germination parameters of sunflower seedlings²⁵. Few reports on the effects of Fe-NPs in plants are available, Shankramma et al.²⁸ stated that the research outcomes depend on the nature of the NPs and plant species and are not always consistent with each other. However, the crops in this study showed no negative effects during germination with varying concentrations of Fe₃O₄-NPs. This is due to the reaction of plant growth stimulant microalgae metabolites with FeCL₂¹⁴. This is consistent with the findings of Ilona et al. (2019), who found that Fe₃O₄-NPs at concentrations of 1 mg L⁻¹, 2 mg L⁻¹, and 4 mg L⁻¹ induce low genotoxicity and have a beneficial effect on the growth and development of rocket seedlings, implying that nanoparticles may improve plants’ resistance to environmental stresses²⁹. Polischuk et al. (2019) also noted that Fe₃O₄-NPs treatment increased 10% in seed germination compared to the control, as well as a 25–30% increase in root growth³⁰. On the other hand, González-Melendi et al. (2008) and Zhu et al. (2008) searched into Fe-oxides and found that they are relatively safe for nanoparticle delivery in plants^31,32.

Germination of maize, rice, mustard, greengram and watermelon with different Fe₃O₄-NPs synthesized by different pH. Data are mean ± SD. Each group without sharing letter mean statistical significance (P ≤ 0.05).

Germination characteristics of tested crops (A) corn, (B) rice, (C) mustard, (D) watermelon, (E) greengram.

Percentage of seed germination in vitro condition with different doses of Fe₃O₄-NPs synthesized by pH-12. Data are mean ± SD. Each group without sharing letter mean statistical significance (P ≤ 0.05).

Effect of Fe₃O₄-NPs, bulk FeCl₂.4H₂O (0.1 M), and Gibberellic acid on Vigor index of the tested crops. Values in each bar are represented as mean ± SD.

Qualitative assessment of antifungal activity against Fusarium oxysporum, Fusarium tricinctum, Fusarium maniliforme, Rhizoctonia solani, and Phythium sp. growth were carried out. All tested fungal growth showed inhibition when treated with Fe₃O₄-NPs. Each phytopathogen had an inhibition zone diameter ranging from 10 to 25 mm (Fig. 6). The results clearly demonstrate that iron oxide nanoparticles at the concentrations used in this study (1 mg L⁻¹) showed the inhibition of radial growth of all fungal pathogens tested. The appearance of an inhibition zone on culture media demonstrates the iron oxide nanoparticles’ biocidal activity³³. Additionally, Nehra et al. (2017) demonstrated that iron oxide nanoparticles have antifungal and antibacterial activity. As a result, they concluded that iron oxide nanoparticles can be effectively used as antimicrobial agents³⁴.

Antifungal zone of inhibition by iron oxide (Fe₃O₄) nanoparticles (From left to right) Fusarium oxysporum, Fusarium tricinctum, Phythium sp. Fusarium maniliforme, and Rhizoctonia solani.

Morphological study of Fe₃O₄-NPs was conducted using both the scanning and transmission electron microscopy (Fig. 7). It can be observed in the SEM images that the Chlorella K01 extracts mediated synthesis of Fe₃O₄-NPs in a monodispersed form that are spherical in shape, and which are well separated without any evident aggregation (Fig. 7a,b). This excellent dispersion and spherical morphology of the NPs can be ascribed to the outstanding capping ability of the biochemical in the extracts of Chlorella K01. TEM analysis revealed the size and morphology of the synthesized NPs. The spherical biofabricated Fe₃O₄-NPs were in the range of approximately 50 to 100 nm in size (Fig. 7c,d).

SEM (“a” and “b”) and TEM (“c” and “d”) images of the as prepared Fe₃O₄-NPs using Chlorella K01 extracts as capping and reducing gents.

By employing an X-ray diffraction technique, we were able to determine the crystalline structure of the biofabricated Fe₃O₄-NPs. The XRD profile of the biofabricated Fe₃O₄-NPs is illustrated in Fig. 8. The XRD pattern of the Fe₃O₄-NPs depict various spectral peaks at 2-theta = 31.2°, 33.6°, 36.2°, 46°, 54.8°, 57.1°, and 64°, that can be ascribed to their relevant indices and diffraction planes (111), (220), (311), (400), (422), (511), and (440), respectively. The diffraction planes of the current Fe₃O₄-NPs are very much similar to that reported earlier for Fe₃O₄-NPs³⁵.

XRD pattern of the biosynthesized Fe₃O₄-NPs.

X-ray photoelectron spectroscopy was conducted to confirm the synthesis of Fe₃O₄ nanoparticles and to analyze their oxidation states (Fig. 9). The XPS survey spectrum of Fe₃O₄ nanoparticles synthesized by Chlorella K01 extracts, showed the presence of Fe, O, C, and N (Fig. 9A). This full-scan resulted in to the high resolution subsequent spectra acquisition. The data was fitted, using the “XPSPEAK4.1” program available at https://xpspeak.software.informer.com/4.1. The two peaks in Fe2p for Fe₃O₄-K01 extract sample, at approximately 714 eV and 723.5 eV can be ascribed to the binding energies of Fe³⁺ oxidation state of iron while the peak around 710.6 eV can be attributed to the binding energy of Fe²⁺ (Fig. 9B). Almost similar peak areas of the two Fe³⁺ peaks in the Fe2p XPS spectrum, indicates the synthesis of magnetite nanoparticles (Fe₃O₄)³⁶. The deconvolution of the O1s spectrum exhibited valuable information regarding the chemical states of oxygen linkage in the as prepared Fe₃O₄ (Fig. 9C). One peak at 531 eV is associated with the lattice oxygen (O in Fe–O–H), whereas the second peak at 530.1 eV can be attributed to the oxygen in Fe–O. The third peak illustrated in the O1s spectrum with binding energy of 529.6 eV is comparable to that observed in the literature as X = O (where X can be any active component in the biomolecule) and may be a by-product generated during the biosynthesis of Fe₃O₄ nanoparticles using Chlorella K01 extracts. These binding energies are due to the interactions between the Fe and the oxygen containing functional groups in the biological system. The bioactive materials containing these functionalities can react metal ions through ion exchange reactions, hence producing metal oxides (Fe₃O₄ in this case) nanoparticles. For the C1s XPS spectrum, the existence of peaks at binding energies 284.6 eV, 285 eV, 286.4 eV and 288.5 eV can be attributed to (C–C), (C–N), (C–O) and (C=O) linkages, respectively (Fig. 9D). Furthermore, the N1s XPS spectrum of Fe₃O₄-K01 can be deconvoluted into two component peaks, namely pyrrolic nitrogen (400.1 eV) and nitrogen associated with carbon in the form of C–N at binding energy 399.5 eV (Fig. 9E). Similar findings have also been reported by Khan et al.³⁷.

X-ray photoelectron spectra (XPS) of Fe₃O₄-K01 extracts. Survey scan XPS spectrum (A), Fe 2p (B) O1s (C), C1s (D) and N1s (E) spectra of the K01 extracts based synthesis of Fe₃O₄ nanoparticles. The black lines (little noisy) denote experimental raw data, the overlaid red line is the deconvoluted form of raw data (sum of all fitting data), the baseline is presented by blue color and other color lines are the fitting data.

The FTIR analysis was carried out in order to classify the functional groups in biomolecules extracted from Chlorella K01 that were utilized for the reduction and capping of the Fe₃O₄-NPs (Fig. 10). The spectral bands at wave number 3710 cm⁻¹ and 2815 cm⁻¹ are more prominent as compared to others at 2255 cm⁻¹, 2550 cm⁻¹, and 3410 cm⁻¹. The active and prominent band at 3710 cm⁻¹ confirmed the presence of O—H stretching indicating the polyphenolic group. The second dominant band at 2815 cm⁻¹ can be ascribed to C—H stretching of aldehyde functional group. The other bands observed at 2255 cm⁻¹, 2550 cm⁻¹, and 3410 cm⁻¹ correspond to C≡C, S—H, and N—H stretching vibrations which illustrate the presence of alkyne, thiol and primary amine functional groups, respectively. These results suggested that the extract of Chlorella K01 containing afore mentioned functional groups are involved in the reduction of FeCl₂ in Fe₃O₄-NPs. Various earlier reports are in line with the current findings where they have reported amine, hydroxyl, carboxylic and phosphate functional groups in algal extracts are involved in the biofabrication of metal nanoparticles³⁸.

FTIR spectral analysis of the biofabricated Fe₃O₄-NPs.

The zeta potential of the biofabricated Fe₃O₄-NPs was observed as a sharp single peak in the range of − 48 and 0 mV, having a maximum intensity at − 25.8 mV (Fig. 11a). This suggested that the surface of Fe₃O₄-NPs consists of negatively charged moieties that expanded in the medium. The dispersion of the NPs might be due to repulsive nature of the negative values which also suggested stability of the Fe₃O₄-NPs. Lower values of zeta potential depict minimum or no flocculation and reduced tendency towards assembly.

Zeta potential (a) and dynamic light scattering (b) of the biofabricated Fe₃O₄-NPs, depicting the surface charge values and particle size distribution, respectively.

DLS analysis reveals the particle size and distribution in the materials. DLS of the biofabricated Fe₃O₄-NPs illustrate a particle size range of 20 to 200 nm (Fig. 11b). It was found that the average particle size of the Fe₃O₄-NPs was 76.5 nm. The particle size and distribution identified via DLS analysis is consistent with that measured by SEM and TEM analysis.