Algae-Mediated Synthesis and Structural Characteriza6on of Iron Nanopar6cles using Galaxaura rugosa Seaweed

: A simple reduc-on method has been used for successfully synthesizing Galaxaura rugosa -mediated iron nanopar-cles (FeNPs). The FeNPs were characterized using UV-visible spectroscopy, X-ray diﬀrac-on analysis (XRD), Transmission electron microscopy (TEM), Selected area electron diﬀrac-on (SAED), Scanning electron microscopy (SEM), Energy dispersive analysis of X-rays (EDAX), Zeta poten-al, and Fourier transform infrared spectroscopy (FTIR). The early forma-on of FeNPs is supported by rapid color change from yellow to dark brown and UV-visible absorp-on peaks at 327 nm. The XRD paRern and SAED analyses show a crystalline nature of FeNPs. Biogenic FeNPs were found to be spherical with mean diameter sizes varying from 14.4 to 17.2 nm by TEM image. The high abundance of FeNPs produced is visible in SEM, and the par-cles are in aggregates. The biosynthesized FeNPs had a nega-ve surface charge with zeta poten-al values of -38.4 mV. According to FTIR analysis, func-onal groups play a signiﬁcant role in the bioreduc-on of iron ions and the stability of FeNPs. This method is straighXorward in applica-on and could be performed in eco-friendly projects.


INTRODUCTION
Nanopar(cles (NPs) due to their small size, high surface area, crystal form, unique network order, and high reac(vity are considered a novel technology (He et al., 2023).Metallic NPs have unique characteris(cs such as surface Plasmon resonance, extremely small size, and large surface-to-volume ra(o (Sharma et al., 2016).They also exhibit magne(c and op(cal polarizability, as well as electrical and thermal conduc(vity (Yaqoob et al., 2020).These proper(es have driven significant advancements in both scien(fic research and industrial applica(ons.Among all metal NPs, iron nanopar(cles (FeNPs) are of much interest to the scien(fic community because of their high magne(c nature, high surface area, and electrical and thermal conduc(vity (Fahmy et al., 2018).FeNPs are also characterized by their low toxicity and simple separa(on methodology (Ali and El-Shehawy, 2023).Several methods exist for the fabrica(on of NPs including physical, chemical, and biological methods.Biological ones are generally cost-effec(ve, nontoxic, scalable, and eco-friendly (El Shehawy et al., 2023b).Among the biological materials, algae are called 'bionanofactories' because both the live and dead dried biomasses were used for the synthesis of metallic NPs (Davis et al., 1998).The key merits of FeNPs green synthesis using algae seaweeds including the algae culturing is rela(vely convenient and easy to handle, synthesis at low temperatures with greater energy efficiency, less toxicity, high-yielding, low-cost technology, and safety to the environment (Rajeshkumar et al., 2017;El Shehawy et al., 2023a).Among seaweeds, the red seaweed Galaxaura rugosa is found in tropical to subtropical marine environments where its presence is indica(ve of the ecosystem's overall health (El-Tabakh et al., 2023).G. rugosa extract contains bioac(ve substances including; polysaccharides, pep(des, and pigments that are efficient in reducing metals to form nanometals (Arguelles, 2022;Mondal et al., 2023).Therefore, the primary objec(ve of this research is to synthesize iron nanopar(cles u(lizing Galaxaura rugosa marine algal extract and subsequently conduct a comprehensive characteriza(on of the produced nanopar(cles.

Algal sampling and prepara.on of algal extract
The seaweed samples were selected and picked from Zaafarana Beach located at the Gulf of Suez, the Red Sea coast of Egypt; 82 km south of Al-Ain EL-Sokhna (29.06"N and 32.43" E) in June 2021.It was iden(fied according to Aleem (1993).The algal extract was prepared by soaking 5 g of dry algal powder in 100 ml of deionized water for 24 hours with frequent shaking then filtered through Whatman Filter paper No.1.The supernatant was used to prepare FeNPs.

FeNPs phyco-synthesis
The established protocol reported by Mahdavi et al. (2013b) was applied to synthesize FeNPs by mixing the algal extract with 0.1 M FeCl3 solu(on in a volume ra(o 1:1, s(rring for one hour, and lej at room temperature for another 30 min.The synthesized FeNPs were collected by centrifuga(on, washed with ethanol, and then washed several (mes with deionized water for characteriza(on.

Characteriza.on of FeNPs
Characteriza(on of the phyco-synthesized FeNPs was carried out by several processes.The UV-visible absorp(on spectra in the 200-600 nm wavelength range using UV-visible absorp(on spectroscopy (Uni cam UV-VIS.Spectrometer UV2, U.S.A) (Basavaraja et al., 2011).FT-IR of the studied seaweed and FeNPs were measured over the range of 400-4000 cm −1 on a Bruker, Tensor 37 FT-IR spectrophotometer (Wen et al., 1996).The size and shape of FeNPs were visualized by TEM (JEOLJEM-2100, U.S.A).The crystalline structure was described through the crystallographic experimental technique performed inside the TEM by SAED (Ruud et al., 1976).Also, the FEI-TITAN 80-300 kV SEM was used to study the morphological structure of the FeNPs.The elements presented in FeNPs were determined by EDX integrated into the SEM.XRD analysis was obtained by a DX-1000 X-ray powder diffractometer range at 40 kV and 30 mA, in the 2θ range of 10°-90° (Sharma et al., 2012).

Collec.on and taxonomic descrip.on of seaweed sample
Figure 1(a) shows the study area where the algal samples were selected and picked.Algae are iden(fied as red algae (Rhodophyta) namely; Galaxaura rugosa (J.Ellis & Solander) (Figure 1b) (Aleem, 1993).

Biosynthesis and UV-visible analysis of FeNPs
UV-visible absorp(on spectra were used to confirm the phyco-synthesis of FeNPs.The maximum absorp(on peak of FeNPs was observed at 327 nm (Figure 2a), while the algal extract shows a major absorbance peak in the UV region at 230 nm (Figure 2b).This result is similar to what was reported in earlier studies by Saranya et al. (2017) that exhibited a characteris(c absorp(on peak of iron nanopar(cles between 250-350 nm.The inset photo shows the preliminary indica(on for the forma(on of FeNPs where the color of the algal extract changes from pale yellow to yellowish brown.The color change is due to the excita(on of the surface plasmon resonance in the metal nanopar(cles (Mahdavi et al. 2013a).

FT-IR analysis of FeNPs
FT-IR analysis is known as a fundamental applying technique of func(onal group detec(on and it has been used in this study to support the determina(on of the reduc(on or forma(on of the func(onal groups during the bio-reduc(on, capping, and stabiliza(on processes of the phyco-synthesized FeNPs (Rehman and Bonfield, 1997;Malik et al., 2021).FTIR of the aqueous extracts of G. rugosa (Figure 3) exhibit a dis(nc(ve peak at 3421 cm -1 , asributed to the O-H stretching vibra(ons within polyphenols and OH groups of sugar rings (Balaraman et al., 2020).As shown in Figure 3, the presence and shijing peaks in the FeNPs indicate interac(on among the func(onal groups of the extract and the iron salt precursor (Rasheed et al., 2018).The absorp(on peaks that appeared at 1598 cm −1 may be due to the vibra(ons in the C=O bond stretching within the aroma(c rings of different phenolic compounds, including polyphenols and flavonoids that are present in the extract (Venkateswarlu et al., 2019).The FTIR spectra of the FeNPs showed a minor shij with slight changes, indica(ng that the main biomolecules present in extracts were capped to the FeNPs surface El-Kassas and El Komi (2014).

TEM Analysis and SAED paIern of FeNPs
To further study the microstructure, morphology, and distribu(on of the phyco-synthesized FeNPs, TEM was used (Kouhbanani et al., 2019).The TEM image of the phyco-synthesized FeNPs presented in spherical shapes with approximately poly-dispersed par(cles with a size range smaller than 50 nm in size (Figure 4a).This coincides well with previous results (Sathishkumar et al., 2018).Compared to previous similar studies, the nanopar(cles in this study exhibited a smaller size compared to those reported in previous research.While other iron nanopar(cles typically range from 50 to 100 nm (Ting and Chin, 2020).This can be related to pH, temperature, incuba(on (me, type, and concentra(on of precursors that are influencing the biosynthesis of nanopar(cles at varying levels (Patra and Baek, 2014).Analysis of the SAED pasern (Figure 4b) confirms the characteris(cs of polycrystalline material rings that are asributed to crystalline FeNPs (Łukowiec and Radoń, 2020).

EDX and SEM Analysis of FeNPs
The elemental composi(on of FeNPs was ascertained through EDX analysis.EDX spectral analysis of FeNPs (Figure 5) shows strong peaks observed at 0.5, 6.0, and 6.6 keV confirming the presence of FeNPs (Salem et al., 2019).SEM analyses (inset in Figure 5) strongly confirm the spherically shaped, highly distributed, and polydisperse of the phyco-synthesized FeNPs, this  is well in line with previous observa(ons (Salem et al., 2019).

CONCLUSIONS
In the current research work, a green synthesis approach was used to synthesize FeNPs that were cost-effec(ve and environmentally friendly.The aqueous extract of Galaxaura rugosa encompasses sufficient biomolecules, which serve as both an an(-agglomera(on and reducing agent.UV-Vis, FT-IR, TEM, SAED, EDX, SEM, XRD, and Zeta poten(al    analysis confirmed FeNPs biosynthesis.The knowledge of the present study as a green method of synthesizing FeNPs could also be extended to fabricate other, industrially important metal oxides which could serve as an economical source of biosynthesis of nanopar(cles.

Figure 2 .
Figure 2. UV-vis spectra of (a) the phyco-synthesized FeNPs and (b) Galaxaura rugosa marine algal extract, inset shows extract before and a^er exposure to Fe solu-on.