Extracellular Synthesis of Iron Oxide Nanoparticles Using an Extract of Bacillus circulans : Characterization and In Vitro Antioxidant Activity

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Introduction
Nanotechnology is a rapidly evolving area of technology that has recently transformed health, food, agriculture, and industry.Nanotechnology is a vibrant feld of science with utile products, including nanorods, nanotubes, and nanoparticles of varying dimensions.It deals with particles of minimal sizes ranging from 1 nm to 100 nm.Te nanoparticles possess coveted properties like colloidal stability and optical and magnetic properties, making them a cut above bulk materials [1].
Metal nanoparticles ofer promising applications in catalysis, drug delivery, cancer therapy, pollutant removal, water treatment, pigments, cosmetics, and medicine.Scientists have synthesized many metal-based nanoparticles, including silver, gold, zinc, iron, selenium, copper, and chromium nanoparticles.Among these metal nanoparticles, magnetic iron oxide nanoparticles hold prime importance in biomedical applications due to their physiochemical and magnetic properties.Te IONPs exist in diferent polymorphic forms such as α-Fe 2 O 3 (hematite), β-Fe 2 O 3 , c-Fe 2 O 3 (maghemite), FeO (wustite), and Fe 3 O 4 (magnetite).Magnetite, hematite, and maghemite are the most prevalent and valuable iron oxide nanoparticles [2].Te separation of a catalyst from the liquid phase can be performed using magnetic IONPs and applying an external magnetic feld by small magnets [3].Te magnetic properties of IONPs are benefcial in immobilizing diamagnetic materials such as metal nanoparticles, organocatalysts, and enzymes acting as catalyst support [4].Moreover, magnetic IONPs are used as contrast agents in MRI and hypothermia treatment [5].
Te synthetic pathways are crucial for the properties of the IONPs.Te properties of NPs, such as size, crystal structure, and magnetic and surface properties, are afected mainly by their synthesis route.Te synthesis of IONPs is primarily carried by "top-down" and "bottom-up" approaches.Te "top-down" techniques involve breaking bulk materials into nanoparticles using physical methods such as sonication, laser ablation, mechanical milling, high-velocity deformation, and physical vapor deposition [6].Tese techniques are expensive, time-consuming, and lack the biocompatibility of nanoparticles.Te "bottomup" approaches build nanoparticles from molecular or atomic particles through chemical or biological methods.Several methods to synthesize IONPs rely on chemical processes, which require expensive chemicals and equipment and produce nanoparticles with toxic impurities [7].Te metal-reducing ability of specifc metabolites from biological systems such as plants, fungi, and microorganisms inspires biological approaches.Biosynthetic pathways produce nanoparticles using proteins, fatty acids, enzymes, peptides, and favonoids.Te fast growth of microorganisms makes large-scale production of nanoparticles possible [8].Te nanoparticles synthesized using plants or microorganisms are biocompatible, do not contain any toxic impurities, and are safe for medical applications [9].Moreover, biogenic synthesis reduces energy consumption as all reactions occur at room temperature.Terefore, biological methods to fabricate IONPs are successful tools for producing nanoparticles in an ecofriendly manner.
Microbial synthesis of IONPs has emerged as a costefective and sustainable method.Many researchers have used bacteria to fabricate magnetic IONPs with fne control on size, surface, and thermal properties.Bacterial synthesis of IONPs can be carried out intracellular or extracellular.Hassan et al. and Fani et al. reported the extracellular synthesis of IONPs using E. coli and Lactobacillus fermentum, respectively [10,11].Similarly, extracellular synthesis of IONPs by Bacillus subtilis and B. cereus also has been reported [12,13].Mukherjee reported intracellular synthesis of copper, silver, and iron nanoparticles using living cells of Stenotrphomonas maltophilia and Microbacterium marinilacus [14].Intracellular synthesis involves binding metal ions or metal oxide ions to the bacteria's cell wall and difusion into the cell, following the enzymatic reduction into IONPs.In extracellular synthesis, the proteins or enzymes in bacterial extract reduce metal ions into IONPs [15].
However, the bacterial systems produce IONPs much slower, and the incubation period for synthesizing IONPs ranges from 1 day to 10 weeks [16].Terefore, it is necessary to look for new bacterial strains that can produce nanoparticles more quickly while retaining their critical physiochemical properties.Te current study introduces a quick, efcient, and easy strategy for synthesizing IONPs using the supernatant of Bacillus circulans.Te in vitro antioxidant activity of as-synthesized and calcined IONPs has been measured by DPPH and ABTS assays [17].IONPs were characterized using UV-visible spectroscopy, FT-IR spectroscopy, FE-SEM, XRD, and EDX [18,19].

Growth of Bacterial Cultures.
Te bacterial colony identifed as Bacillus circulans was procured from the Department of Zoology microbiology laboratory, Government College University Lahore, Pakistan.Te solution of nutrient agar broth in a conical fask containing a loopful of Bacillus circulans was incubated overnight at 37 °C and 85 rpm in a shaker incubator for maximum growth of bacteria.

Extraction of Bacterial Supernatant.
Te bacterial cultures grown overnight were centrifuged at 6000 rpm in 15 ml falcon tubes for 5 minutes.Te upper layer of supernatant was isolated into a conical fask.Te bacterial pellets were discarded, and the bacterial extract was used to synthesize IONPs.

Biosynthesis of IONPs. Te extracellular biosynthesis of
IONPs was carried out by mixing the 25 ml of 0.1 M solutions of FeSO 4 .7H 2 O and 25 ml bacterial supernatant and shaking the mixture at 80 rpm for fve minutes.Te mixture containing the bacterial extract and IONPs was centrifuged at 5000 rpm for fve minutes to isolate nanoparticles.After washing with distilled water, the nanoparticles were dried in an oven for 24 hours at 60 °C and stored to characterize and evaluate the antioxidant activity.

Optimization of Conditions.
Te condition optimization for synthesizing IONPs was carried out by changing the concentrations of salt solution, pH, and temperature.Te 0.1 M, 0.3 M, 0.5 M, and 1 M salt solutions were used at pH 7 and 40 °C.Te optimum temperature was found by measuring the yield of IONPs at various temperature conditions of 50 °C, 70 °C, and 90 °C, keeping the concentration of the salt solution and pH constant.Similarly, optimum pH was found by determining the yield of IONPs at various pH values of 5, 6, 7, 8, and 9, keeping concentration and temperature constant.

Characterization of IONPs.
Te IONPs were characterized using the UV-visible spectrophotometer, model UV-2300, Techom, with wavelength scanning in the 200-800 nm range; the absorption spectrum associated with surface plasmon resonance of the IONPs was recorded.Te surface functional groups and bonds in the IONPs were determined using a Fourier transform infrared (FT-IR) spectrophotometer, IRPrestige-21.Te FT-IR spectrum of the bacterial supernatant was recorded to fnd the potential functional groups responsible for the synthesis of IONPs.Te surface roughness and morphology of the IONPs were determined using the FE-SEM (NovaNano SEM 450) at 10kV.Te quantitative compositional analysis of the nanoparticles was performed using FE-SEM (NovaNano SEM) with an Oxford EDX detector.An advanced XRD system, Bruker D2-Phaser, was used to identify the crystal structure of the IONPs.Te Xpert Highscore Plus was used for peak indexing and phase identifcation.

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Journal of Chemistry 2.6.Evaluation of Antioxidant Activity.Te antioxidant activity of the IONPs was determined using the ABTS assay and DPPH assay.

ABTS Assay.
ABTS radical cation-based assays are abundant in antioxidant activity tests [20].In this study, the antioxidant activity of IONPs was evaluated following the modifed method of Arnao et al. [21].10 µL of 1 mg/ml of IONPs was added to 2.99 ml of ABTS •+ solution and kept in the dark for 8 minutes.Te absorbance of ABTS •+ solution at 734 nm before and after adding NPs solution was recorded using a UV-visible spectrophotometer.Te percentage inhibition of ABTS •+ was calculated using the following equation: where A 0 is the maximum absorbance of the standard ABTS •+ solution and A is the maximum absorbance of the ABTS •+ solution following the addition of IONP.
2.8.DPPH Assay.DPPH assay is a hydrogen transfer-based antioxidant activity assay.It is a stable, synthetic radical and is widely used in antioxidant assays.Te antioxidant activity of IONPs was determined by DPPH assay following the modifed Brand-Williams method.In the DPPH assay, 10 µL of biologically synthesized IONPs was mixed with a 2.5 ml solution of DPPH and shaken vigorously.Te maximum absorbance of the DPPH solution at 517 nm and the decrease in absorbance on adding NPs solution were recorded by a UV-visible spectrophotometer.Te decrease in absorption of the DPPH solution at 517 nm was recorded.Te antioxidant activity was measured by calculating the percentage scavenging of DPPH using the following equation: Here, A and A 0 are the absorbances of the DPPH solution containing IONPs ad standard DPPH solution, respectively.

Results and Discussion
3.1.Biosynthesis of IONPs.Te IONPs were synthesized extracellularly using a supernatant of Bacillus circulans.Te yellow color of the bacterial supernatant changed to greenish black immediately after adding a solution of FeSO 4 .7H 2 O, indicating the formation of IONPs.Te color change in the appearance of IONPs is due to the surface plasmon resonance of the nanoparticles.Te color change is depicted in Figure 1.
Te possible mechanism involved in the extracellular synthesis of IONPs using bacterial extract is the bioreduction of Fe 2+ by proteins in the bacterial supernatant [22].Te frst step in this process is the reduction of divalent iron ions (Fe 2+ ) in the precursor salt (FeSO 4 .7H 2 O) to metal form (Fe 0 ).Te proteins in the bacterial supernatant may act as reducing agents.Te FT-IR of the bacterial supernatant shows the presence of the -OH group, which is electron-rich and can donate electrons to Fe 2+ , reducing it to the metal form.Te second step encompasses the oxidation of metallic iron atoms to iron oxide.Te fnal step is the stabilization of IONPs by biomolecules in the bacterial supernatant.FT-IR spectrum of as-synthesized IONPs shows the presence of the -OH group on the surface of nanoparticles, showing that it stabilizes IONPs.

Optimization of Conditions.
Te yield of IONPs in grams at various temperatures, pH, and concentration conditions was determined.It was observed that maximum IONPs were formed at 40 °C and pH 7. At this temperature and pH, the maximum growth of bacteria occurred.Te bacterial extract contained maximum biomolecules, and increasing the concentration of salt solution increased the yield.Te salt concentration is directly related to the yield of IONPs because of increasing metal ions (Figure 2(a)).Te bacteria showed no growth at pH 5, and at pH 6, the yield was low.Te highly acidic or basic conditions retard the growth of bacteria which in turn produce little metabolites, and the yield of nanoparticles also decreases (Figure 2(c)).Te efect of temperature had a variable efect showing a maximum yield at 40 °C and then a decrease in yield at 50 °C.Te reaction speed was more signifcant at higher temperatures, and an intense color change was observed; however, the yield was low.Te decrease in yield at 50 °C and then increase up to 70 °C indicates the presence of more than one metabolite in bacterial extract responsible for producing nanoparticles.Some metabolites could not tolerate temperatures higher than 40 °C and start denaturing.Te other metabolites tolerated temperatures even higher than 50 °C and the yield of IONPs increased.At 90 °C, all the metabolites start denaturing, and the yield of nanoparticles drops (Figure 2(b)).

Characterization of IONPs.
Te fabrication of IONPs was further evaluated using various characterization techniques such as UV-visible spectroscopy, FTIR spectroscopy, FE-SEM, EDX, and XRD.Te UV-visible scan of the 5% diluted solution of nanoparticles was performed in the 200-800 nm range.

UV-Visible Spectroscopic Analysis.
Te UV-visible spectrum of the as-synthesized IONPs shows maximum absorption at 225 nm within the surface plasmon resonance range of the IONPs (Figure 3).Te very intense peak depicts the fabrication of many small-sized IONPs.Te peak in the UV-visible spectrum corresponds to the surface plasmon resonance (SPR) of maghemite (c-Fe 2 O 3 ) NPs. Chauhan and Upadhyay have reported similar absorption by iron oxide nanoparticles produced using an extract of Lawsonia inermis.Tey observed an absorption peak around 224 nm [23].Te optical band gap of as-synthesized IONPs calculated using a Tauc plot is 5.39 eV (Figure 4).Te higher value of the band gap indicates small-sized nanoparticles.Small nanoparticles have fewer atoms and poor orbital overlap, increasing the band gap.[24].From this correlation analysis, it can be inferred that the biomolecules present in the bacterial supernatant in the current study are proteins.

Journal of Chemistry
FT-IR spectrum of the as-synthesized IONPs reveals the presence of surface O-H groups as indicated by a peak at 3244 cm −1 .Additionally, the peak at 995 cm −1 corresponds to C-H bending, indicating the presence of biomolecules acting as capping agents to stabilize the IONPs.Te peak at 472 cm −1 corresponds to Fe-O stretching vibrations, characteristic of IONPs.Finally, the 1632 cm −1 and 1431 cm −1 peaks correspond to C�O and C-O stretching vibrations, respectively (Figure 5(a)).Te peaks corresponding to C-H bending and C�O and C-O stretching vibrations confrm that the biomolecules act as capping agents and stabilize the IONPs.Te surface of IONPs is covered with biomolecules such as amino acids or peptides.
3.6.SEM Analysis.Te SEM analysis of morphological features revealed the spherical, uniform, and granular morphology of the as-synthesized IONPs.Te nanoparticles were agglomerated, forming irregularly shaped clusters.Te cluster formed due to the capping agents covering the nanoparticles' surface.Te mean diameter of IONPs determined using SEM was 18.34 nm.Te SEM images highlighted the surface roughness of as-synthesized IONPs (Figure 6). Figure 7 shows the uniform size distribution of the nanoparticles.622), (421), (311), and (111), respectively (Figure 9).Te relative intensities and positions of these peaks closely matched the maghemite crystal structure (c-Fe 2 O 3 ) corresponding to ICCD card number 39-1346, confrming that the major phase of the IONPs is the cubic crystalline form of maghemite (c-Fe 2 O 3 ).Additionally, the peaks positioned at 45.38 °, 31.66 °, and 28.64 °are indexed to (160), (102), and (230), respectively, and closely matched the miller indices given in ICCD card no.33-0665 confrms the presence of the orthorhombic crystalline phase of iron oxide carbonate.Te peaks other than IONPs appear due to the presence of biomolecules contributing to the production of iron oxide carbonate.Te as-synthesized IONPs had an average crystallite size of 13.84 nm, which was calculated using the following equation:   where D � crystallite size, K � Scherrer constant, B � FWHM values of the peaks, λ � X-ray wavelength, and θ � Bragg's angle.
Te phase identifcation of the calcined IONPs was performed using an XRD pattern.Te peak analysis revealed that the calcined IONPs have hematite and iron oxide carbonate phases.Te difraction peaks positioned at 75.47 °, 54.087 °, 49.625 °, 35.903 °, 33.37 °, and 24.5 °are indexed to (220), ( 116), (024), ( 110), (104), and (012), respectively (Figure 10).Tese peaks are closely related to the peak position and intensities of rhombohedral hematite (α-Fe 2 O 3 ) corresponding to ICCD card number 33-0664.Te higher antioxidant activity of as-synthesized IONPs than calcined IONPs is due to the presence of biomolecules covering the surface.Te surface of as-synthesized IONPs is covered with biomolecules rich in hydrogen and electrons which are responsible for higher antioxidant activities by hydrogen transfer or electron transfer mechanisms.During calcination, the biomolecules on the surface of NPs decomposed and the antioxidant activity of calcined IONPs decreased.

Conclusion
Te iron oxide nanoparticles (IONPs) were produced using Bacillus circulans supernatant and were found to be spherical with a mean diameter of 18.32 nm and were agglomerated, forming irregular-shaped clusters.Te nanoparticles were analyzed using various techniques such as UV-visible spectroscopy, FT-IR spectroscopy, SEM, EDX, and XRD.Te majority of the as-synthesized IONPs were found to be maghemite (c-Fe 2 O 3 ), while the calcined IONPs were primarily hematite (α-Fe 2 O 3 ).Te optical band gap of IONPs was found to be 5.39 ev showing the formation of ultrasmall nanoparticles.Te as-synthesized IONPs have shown more signifcant ABTS radical cation inhibition and DPPH scavenging activity than calcined IONPs.Teir use can be extended to degrading dyes and removing heavy metals from wastewater.

Figure 1 :
Figure 1: (a) Bacterial supernatant and (b) color change on formation of IONPs.

3. 7 .
EDX Analysis.Te EDX analysis revealed the elemental composition of as-synthesized IONPs.Te EDX spectrum indicates that the IONPs primarily comprise Fe, O, and C. Fe, O, and C percentages are 41.74%, 27.36%, and 11.39%, respectively (Table1).It is inferred from the relative weight percentage of Fe and O that IONPs are chemically Fe 2 O 3 .Te C signal in the spectrum indicates the presence of biomolecules stabilizing the nanoparticles (Figure8).

Figure 3 :
Figure 3: UV-visible spectrum of IONPs synthesized in this study.

3. 9 .
In Vitro Antioxidant Activity 3.9.1.ABTS Assay.Te extent of decoloration of the bluegreen solution of ABTS •+ is a measure of the antioxidant activity of the IONPs[25,26].Te in vitro antioxidant activity by ABTS assay showed the higher antioxidant activity of as-synthesized IONPs than the calcined IONPs.Te antioxidant activity of as-synthesized and calcined IONPs was expressed as percentage inhibition of ABTS •+ .As-synthesized IONPs with 39.44% of percentage ABTS •+ inhibition were more efective antioxidants than calcined IONPs with 35.04% of percentage ABTS •+ inhibition.DPPH Assay: Te DPPH is a stable, synthetic radical widely used in antioxidant activity assays.Te color of the DPPH radical solution is deep purple and fades away upon adding IONPs.Te percentage of DPPH scavenging measures the antioxidant activity of the nanoparticles.Te DPPH assay of the IONPs synthesized in this study revealed that assynthesized IONPs are more signifcant antioxidants with a percentage DPPH scavenging of 35.44% compared to calcined IONPs having a percentage DPPH scavenging of 26.5%.Te enhanced antioxidant activity of assynthesized IONPs compared to bare IONPs is because of biomolecules on the surface of nanoparticles that act as efective hydrogen sources.Te DPPH scavenging activity of both the as-synthesized and calcined IONPs was less than ABTS •+ inhibition activity, possibly due to diferent mechanisms involved in the free radical scavenging.Te results are shown in Figure 11.
Te peaks at 45.75 °and 31.89°indexed to (160) and (102), respectively, closely matched the peaks of the orthorhombic crystalline phase of iron oxide carbonate according to ICCD card number 33-0665.Te average crystallite size of calcined IONPs calculated using the Scherrer equation was 23.18 nm.