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

The purpose of this research was to determine the efficacy of iron oxide nanoparticles (Fe3O4-NPs) using microalgal products as a plant growth stimulant and antifungal agent. The work was conducted with the phyco-synthesis and characterization of Fe3O4-NPs using 0.1 M ferric/ferrous chloride solution (2:1 ratio; 65 °C) with aqueous extract of the green microalga Chlorella K01. Protein, carbohydrate and polyphenol contents of Chlorella K01 extract were measured. The synthesized microalgal Fe3O4-NPs made a significant contribution to the germination and vigor index of rice, maize, mustard, green grams, and watermelons. Fe3O4-NPs also exhibited antifungal activity against Fusarium oxysporum, Fusarium tricinctum, Fusarium maniliforme, Rhizoctonia solani, and Phythium sp. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) scanning electron microscopy (SEM), transmission electron microscopy (TEM), particle size analysers (PSA), and zeta potential (ZP) measurements were used to characterize these green fabricated magnetite NPs. FTIR analysis showed that the synergy of microalgal proteins, carbohydrtates and polyphenols is responsible for the biofabrication of iron nanoparticles. A spheroid dispersion of biosynthesized Fe3O4-NPs with an average diameter of 76.5 nm was produced in the synthetic process.

www.nature.com/scientificreports/ effective nanomaterial-based fungicides for the control of some plant fungal diseases 17 . They have the potential to be widely used in agriculture as biocontrol agents to promote sustainable agriculture 18 . The environmentally green chemistry approach thus provides a clean, nontoxic, and environmentally friendly method of producing NPs with a wide range of size, morphology, component, and physical and chemical properties 19 . Moreover, metal oxide nanoparticles are stable and are considered to be safe for humans 20 .
The main purpose of this study was to synthesize iron (Fe) nanoparticles based on microalgae for agricultural purposes. Aqueous extracts of Chlorella K01 have been used to biosynthesize environmenta friendly plant growth stimulants and anti-fungal Fe nanoparticles (Fe 3 O 4 -NPs).

Materials and methods
All chemicals used were of analytical reagent grade and purchased from Aladdin, China. Chlorella K01 was gifted by Professor Prezemyslaw Malec and Dr. Jan Burczyk from Jagiellonian University, Krakow, Poland.
Preparation of algal extract and determination of protein, carbohydrate and polyphenol contents. Chlorella K01 was cultivated in fresh KC medium at 25-40 (μE m −2 s −1 ). Start-up seed culture was done in 250 ml sterile flasks having 100 ml KC medium. To obtain biomass, it was subcultured in 500 ml flasks and then larger 1000 ml flasks. The culture was washed with water, freeze dried and stored. To prepare this extract, 100 ml of ultrapure water was heated at 60-70 °C with 0.1 g of algal powder. The raw extract algal was constantly agitated, then filtered and the obtained supernatant was used as the algal bioextract.
The kit #16-6002 Bao Ruyi (Beijing) Biotechnology Co., Ltd used the BCA method to determine protein concentrations (bicinchoninic acid) 21 . The BCA/copper complex absorbance was measured at 562 nm in a UV-Vis spectrophotometer. A 0.5 mg ml −1 bovine serum albumin standard curve was used to calculate protein concentration. The total phenolic content of the extracts calculated by using the Folin-Ciocalteu method 22 , gallic acid was the standard. The carbohydrate content was estimated using the phenol-sulfuric acid method. The absorbance at 490 nm was measured the colored aromatic complex formed between the phenol and the carbohydrate with glucose as a standard. in the ratio 2:3, under four different pH conditions. NaOH was used to adjust the pH to 6, 8, 10, and 12. These reactions were kept at 60-70 °C. The synthesized Fe 3 O 4 -NPs were washed three times with 70% ethanol and dried in a hot air oven for 24 h. These iron oxide NPs were synthesized and stored until further use. The effects of germination on various Fe 3 O 4 -NPs seeds synthesized at different pH levels have been examined as shown in "Seed treatment and in vitro seed germination test" section.

Synthesis of
Seed treatment and in vitro seed germination test. The commercialized seeds (rice, maize, mustard, green grams, and watermelons) were bought from the local Longhua market (20° 2′ 9.672″ N, 110° 20′ 3.2634″ E). All the seeds tested in the research are permitted and legal for trade, commercialization in China. Therefore Specific permission was not needed from the Local Authority.
Germination at various Fe 3 O 4 -NPs concentrations was evaluated to determine plant toxicity, as described by Stampoulis et al. 23 . Firstly, soak the seeds in 0.1% mercuric chloride for 3 min, and then rinse thrice with sterilized distilled water. The aseptic seeds were soaked in solution containing (synthesized nanoparticles 1 mg ml −1 , 5 mg ml −1 , 7 mg ml −1 , 10 mg ml −1 , bulk FeCl 2 ·4H 2 O (0.1 M) Gibberellic acid (GA) 15 mg ml −1 ) for 1 h and agitated at 100 rpm. The control was sterilized and treated with distilled water. Afterwards, all of the seeds were transferred to plates that contained two layers of wetted filter papers that were carefully rolled (25 seeds per plate) and incubated at 25 °C under a 16:8 (light: dark) cycle for 7 days. The germination percentage was calculated using normal seedlings. Ten seedlings from each replicate were chosen at random to be measured for shoot and root length. Triplicate experiment was repeated three times. For their comparative study, the vigor index was calculated for seeds throughout germination experiments that exhibit the best responses to Fe 3 O 4 , bulk FeCl 2 ·4H 2 O (0.1 M), Gibberellic acid, and control. A vigor index was calculated as given by Abdul-Baki and Anderson 24 : Anti-fungal activity against phytopathogens. The antifungal activity of Fe 3 O 4 -NPs was examined against the fungal pathogens; Fusarium oxysporum, Fusarium tricinctum, Fusarium maniliforme, Rhizoctonia solani, and Phythium sp. Fungal cultures were incubated in potato dextrose media (PDA) liquid media at 26 °C for 5 days before being cultured on fresh PDA solid media with 1 × 10 7 spores ml −1 . The agar well diffusion method 25  www.nature.com/scientificreports/ captured through the analysis of the prepared grids on an AMT camera system. The particle size was also determined by TEM using an Image Analyser System (IAS). The surface features of Fe 3 O 4 -NPs deposited on a graphite grid were examined using a SEM (model S360 brand SEM-Leica Cambridge, Cambridge, UK).
X-ray diffraction (XRD) and X-ray photoelectron spectroscopy. Cu Kα radiation was used to generate the x-ray diffraction (XRD) pattern, which was recorded using an X-ray diffractometer (Powder X-ray-D8 advanced diffractometer, Burker) from 5_ to 100_ 2θ angles at 40 kV and 30 mA. The exposure time was 300 s. Besides, X-ray photoelectron spectroscopy (Thermo Scientific ESCALAB 250) was used to further analyse the chemical composition and binding energies of the as prepared Fe 3 O 4 nanoparticles 26 .
Fourier transform infrared (FTIR) spectral analyses. The Fe 3 O 4 -NPs colloid was biosynthesized and centrifuged at 10,000 g for 15 min after being lyophilized and grinded with potassium bromide (KBr) powder for FTIR measurements. The spectrum was captured in the 500-4000 cm -1 range using a Bruker, TGA-IR, TENSOR 27 spectrometer in diffuse reflectance mode with a resolution of 4 cm. In accordance with previously published information, spectral absorption bands were identified.

Results and discussion
Iron oxide nanoparticles were successfully prepared using a green approach with microalgal extract in an alkaline medium. Fe 3 O 4 -NPs was created using a microalgal extract in a quick, cost-effective, and environmentally safe way 19  According to the results of the in vitro germination test, Fe 3 O 4 -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 Fe 3 O 4 -NPs concentrations on seed germination, Fe 3 O 4 -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 Fe 3 O 4 -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 seedlings 25 . Few reports on the effects of Fe-NPs in plants are available,    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 Fe 3 O 4 -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 activity 33    www.nature.com/scientificreports/ Morphological study of Fe 3 O 4 -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 Fe 3 O 4 -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 Fe 3 O 4 -NPs were in the range of approximately 50 to 100 nm in size (Fig. 7c,d).
By employing an X-ray diffraction technique, we were able to determine the crystalline structure of the biofabricated X-ray photoelectron spectroscopy was conducted to confirm the synthesis of Fe 3 O 4 nanoparticles and to analyze their oxidation states (Fig. 9). The XPS survey spectrum of Fe 3 O 4 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:// xpspe ak. softw are. infor mer. com/4.1. The two peaks in Fe2p for Fe 3 O 4 -K01 extract sample, at approximately 714 eV and 723.5 eV can be ascribed to the binding energies of Fe 3+ oxidation state of iron while the peak around 710.6 eV can be attributed to the binding energy of Fe 2+ (Fig. 9B). Almost similar peak areas of the two Fe 3+ peaks in the Fe2p XPS spectrum, indicates the synthesis of magnetite nanoparticles (Fe 3 O 4 ) 36 . The deconvolution of the O1s spectrum exhibited valuable information regarding the chemical states of oxygen linkage in the as prepared Fe 3 O 4 (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 Fe 3 O 4 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 (Fe 3 O 4 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 Fe 3 O 4 -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 .
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 Fe 3 O 4 -NPs (Fig. 10)  www.nature.com/scientificreports/ The zeta potential of the biofabricated Fe 3 O 4 -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 Fe 3 O 4 -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 Fe 3 O 4 -NPs. Lower values of zeta potential depict minimum or no flocculation and reduced tendency towards assembly. DLS analysis reveals the particle size and distribution in the materials. DLS of the biofabricated Fe 3 O 4 -NPs illustrate a particle size range of 20 to 200 nm (Fig. 11b). It was found that the average particle size of the Fe 3 O 4 -NPs was 76.5 nm. The particle size and distribution identified via DLS analysis is consistent with that measured by SEM and TEM analysis. www.nature.com/scientificreports/

Conclusions
Iron oxide nanoparticles were generated using microalgal extract as a reducing agent. This method offers a simple yet environmentally friendly approach. When compared to other biological extracts previously reported, Chlorella K01 was found to be more effective, as the NPs obtained with this extract have the lowest zeta potential (− 25.8 mV) and the average particle size of the Fe 3 O 4 -NPs was 76.5 nm. Iron oxide nanoparticles synthesized using this method showed promising plant growth stimulant and antifungal activities against a variety of fungal pathogens, and thus can be used to control a variety of fungal diseases. The Fe 3 O 4 -NPs drastically enhanced rice, corn, mustard, green gram, and watermelon germination.