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Green Synthesis, Characterization and Application of Natural Product Coated Magnetite Nanoparticles for Wastewater Treatment

Adsorption of organic pollutants, toxic metal ions, and removal of harmful bacteria can give us clean and pure drinkable water from wastewater resources. Respective magnetite nanoparticles (MNPs) were synthesized using a cheaper and greener way in an open-air environment with the use of crude latex of Jatropha curcas (JC) and leaf extract of Cinnamomum tamala (CT). Characterization of MNPs had been performed by dynamic light scattering (DLS), Ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, powdered X-ray diffraction (XRD), and field emission scanning electron microscope (FE-SEM). The size ranges of the synthesized MNPs were observed in between 20–42 nm for JC-Fe3O4 and within 26–35 nm for CT-Fe3O4 by FE-SEM images. The effect of synthesized magnetic nanoparticles in wastewater treatment (bacterial portion), dye adsorption, toxic metal removal as well as antibacterial, antioxidant, and cytotoxic activities were studied. This purification will lead to an increase in the resources of pure drinking water in the future.


Introduction
The freshwater scarcity and water pollution problems have been increasingly growing worldwide in the last several years [1]. At present, around 3.1% of deaths happening every year, which is over 1.7 million all over the world, are caused just because of unsafe and lack of reliable sources of drinkable water [2]. It is estimated that more than 57% of the world's population will have difficulties in accessing water throughout the year by 2050 [1]. Water pollution is the principal cause for the lack of suitable drinking water resources. In every growing nation, importance on industry and agricultural evolution leads to contamination of water with harmful organic pollutants and metals like cobalt (Co), copper (Cu),

Preparation of JC-Fe 3 O 4 NPs
One-hundred milliters of DW was added to the mixture of 1.27 g FeCl 2 ·H 2 O and 3.24 g FeCl 3 (anh.), stirred for 30 min, followed by the addition of 125 mL 3% JC latex; 3.2 g NaOH in 50 mL DW was added to the mixture and stirred further for 50 min to complete the reaction. Centrifugate was washed with DW repeatedly two to three times, and then dried in a vacuum desiccator overnight to get the final nanopowder.

Characterization of CT-Fe 3 O 4 and JC-Fe 3 O 4 NPs
DLS (model ZETASIZER Nano Series Nano ZS, Malvern Panalytical, Malvern, UK) technique was used to get a rough idea about the average size and particle size distribution of respective nanoparticles (hydrated sphere). UV-Vis spectroscopy had been done by using Perkin Elmar instrument, Waltham, MA, USA). Clustered metallic oxides were investigated by powdered X-ray diffractometry using Rigaku Miniflex 600 (Japan) equipped with copper X-Ray tube (Cu-Kα1,2 radiation) and NaI (Tl) scintillating detector. The surface morphology of the MNPs was studied using (FE-SEM, MIRA II LMH, Tescan, USA). Fourier-transform infrared spectroscopy (FTIR) was performed by using IR spectrometer (JASCO FT/IR-4600, Tokyo, Japan) with a resolution of 4 cm −1 and the scan range of 650-4000 cm −1 . Magnetization measurements were performed on SQUID magnetometer (Quantum Design MPMS-7) from KBSI (Daejeon, South Korea).

Dye Adsorption Experiment
The dye adsorption study of the CT-Fe 3 O 4 and JC-Fe 3 O 4 NPs was performed by taking a known concentration of methylene blue (MB) (200 mg/L) solution and a definite quantity of MNPs (50 mg) in a conical flask (at pH~7), shaken thoroughly for 120 min at room temperature. At an interval of 20 min, the optical density (OD) was checked for each solution at 660 nm for adsorption kinetic study. For another set of experiments, a series of different initial concentrations (200 mg/L-500 mg/L) of MB were shaken (at pH~7, room temperature) with MNPs (50 mg) for 120 min, and the optical density (OD) was checked for each solution for adsorption isotherm study. While collecting the samples, the nanoparticles were removed by application of an external magnetic field [41].

Toxic Metal Adsorption Experiment (with Concentration)
For metal adsorption study, we chose copper (II) acetate and cobalt (II) chloride as a source for copper (Cu 2+ ) and cobalt (Co 2+ ) ions, respectively. Five different concentrations (400 mg/L, 600 mg/L, 800 mg/L, 1200 mg/L, and 1400 mg/L) of both salt solutions were prepared (pH~7, room temperature). Two milligrams of CT-Fe 3 O 4 NPs and 2.0 mg of JC-Fe 3 O 4 NPs were added separately to each test tube containing 5 mL of each salt solution and were ultrasonicated for 120 min. Next the optical density (at 650 nm and 510 nm for Cu 2+ and Co 2+ solutions respectively) was checked for the solutions [42,43].

Antibacterial Assay for Wastewater Treatment
The sample pond water was collected from Cooch Behar district, West Bengal, India. Three conical flasks, each containing 10 mL of sample pond water, were treated separately with 0.05g CT-Fe 3 O 4 and 0.05 g JC-Fe 3 O 4 NPs alongside a blank experiment, and were shaken for 10 min. Then from the stirred solutions, 100 µL of the mixture was spread on Luria Broth (LB)-agar plates and were incubated for 24 h at 37 • C [44]. For the colony forming units (cfu) calculation, the same process was repeated, using different amounts (0.04 g, 0.02 g and 0.01 g) for each of CT-Fe 3 O 4 and JC-Fe 3 O 4 NPs.

Isolation and Characterization of Bacteria from the Collected Sample
The collected pond water was serially diluted from 10 −1 to 10 −15 times and each dilution was spread on the LB-agar plate using spread plate technique and incubated for 24 h at 37 • C. Next, one bacterial single colony was isolated and subcultured to obtain a pure culture. The pure culture was stored in 3 mL microcentrifuge tube for further assays (Disk Diffusion Test and Minimum inhibitory concentration (MIC) determination). Gram staining was performed on the isolated pure culture. Briefly, loopful of mother culture was taken, and smeared on the glass slide. The smear was air dried and fixed by passing over the flame. A few drops of crystal violet stain were added to the fixed smear and were kept for 1minute. Then a few drops of iodine solution were added. Next, alcohol washing was done for 25-30 s. Furthermore, a few drops of saffranine were added and kept for 1 min. After every step, excess stain was washed off with water and dried in air, and the slides were observed under a microscope.

Disk Diffusion Test
One-hundred microlitres of bacterial suspension (0.5 McFarland Standard) was spread over the surface of the LB-Agar plate and allowed to dry for 10 min. Then two sterile paper disk (5 mm) saturated with 30 µL of CT-Fe 3 O 4 and another with JC-Fe 3 O 4 NPs (200 ppm) were placed in the culture medium. The plates were sealed by parafilm and incubated at 37 • C for 24 h, and the diameter of the resulting inhibition zone in every plate was measured [45,46]. All the experiments were performed in duplicates, and the results were expressed as mean values. NPs were added to 10 columns containing 50 µL of culture medium (LB medium) to maintain the concentration sequence from 4000 ppm to 7.8 ppm. The standardized bacterial culture (1 × 10 5 cfu/mL) was added in each well from columns 1-10. Further, the microlitre-sized wells were incubated at 37 • C for 24 h, and the resulting turbidity was observed. The MIC was determined where there was no visible growth of bacteria detected. For further confirmation, the turbidity was measured by optical density readings at 600 nm with a UV-Vis spectrophotometer [47,48].

Bacteria Culture Preparation
The commercial bacterial stain of E. coli (ATCC 25922) and S. aureus (ATCC 29213) were grown in LB medium for 24 h at 37 • C, and optical density readings were compared to a 0.5 McFarland standard. Both the disk diffusion test and MIC were performed in the same procedure discussed before.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay
Free radical scavenging activity was estimated by the DPPH scavenging assay [49,50]. 10.14 µM solution of DPPH in methanol was added to 500 µL MNPs solutions in methanol (total volume was 5 mL) in different concentrations (0.06 mg/mL, 0.25 mg/mL, 0.57 mg/mL, 1.00 mg/mL, 1.57 mg/mL) and the activity was observed at 517 nm after keeping the solutions in dark for about 30 min. The control sample was prepared without MNPs. Gallic acid was used as a positive control in all cases. The scavenging activity estimation was performed using the formula.
where A o is the absorbance of the control (DPPH + methanol), and A s is the absorbance of the respective sample solutions (sample in methanol + DPPH solution).

Measurement of Cytotoxicity Using MTT Assay
SW480 and HeLa cells (4.5 × 10 3 cells per well) were seeded into 96-well plates. After 24 h, the media was changed to non-serum RPMI 1640, and after a further 24 h, the cells were treated withJC-Fe 3 O 4 and CT-Fe 3 O 4 NPs at different concentrations. The treated cells were incubated for 48 h, washed with cold PBS, and then exposed to MTT with media for 4 h. The media was changed to DMSO, and the dissolved formazan dye was quantified by measuring the absorbance at 540 nm. As a control, untreated cells were examined in the same manner.

Synthesis of Nanoparticles
There are different procedures reported for the synthesis of MNPs by the co-precipitation methods, and most of them require an inert environment and, in some cases, an elevated temperature. Herein our study, we have reported the synthesis of JC-Fe 3 O 4 and CT-Fe 3 O 4 NPs at room temperature without inert gas environment and elevated temperature. In addition, we have replaced the use of ammonium hydroxide solution with NaOH solution and this collective approach makes our synthetic procedure much greener than the other reported methods because of environmentally friendly reagents, low toxicity, and biodegradable products [51]. This collective approach makes our synthetic procedure much greener than the methods reported hitherto in the literature.

Dynamic Light Scattering Experiment
Hydrodynamic size measurements are usually greater than the actual size measurements of the MNPs, which is due to the presence of extra hydrated layers attached on the surface. DLS experiment showed that hydrated MNPs had average sizes of around 154.2 nm for JC-Fe 3 O 4 ( Figure 1a) and around 65 nm for CT-Fe 3 O 4 ( Figure 1b). Hence, from the DLS graphs, we conclude that the formation of nanoparticles was completed in both the cases since the distributions are more even with a narrow distribution range.

UV-Visible Spectroscopy
The UV-Vis spectra ( Figure 2) exhibited the characteristic continuous peak absorption of both the magnetite nanoparticles in the visible range; the absorption range was between 300-800 nm [52]. This confirms the formation of iron oxide nanoparticles. From the appearance of a broadband and the absence of any hump spectra, it may be concluded that not much size difference was present in the synthesized nanoparticles.

FTIR Analysis of JC-Fe3O4 Nanoparticles
Next the FTIR analysis was performed to prove the presence of JC latex as the capping material for the synthesized MNPs. Synthesized JC-Fe3O4 NPs showed a strong absorption band at 1607 cm −1 (stretching vibration of C-N group), which was attributed to the binding of JC latex as the capping agent since this peak was also observed to be a significant peak in case of dried JC latex powder at 1618 cm −1 [37]. Other significant FTIR peaks showed the bands at 3248 cm −1 (N-H stretching for amides), 2923 cm −1 (secondary amine), 1373 cm −1 (-CO-stretching), and 1070 cm −1 (O-H stretching), which clearly proved the presence of protein/peptide on the nanoparticle binding surface. This data also fitted well with the previously reported Jatropha curcas extract capped nanoparticles [53] (Figure

UV-Visible Spectroscopy
The UV-Vis spectra ( Figure 2) exhibited the characteristic continuous peak absorption of both the magnetite nanoparticles in the visible range; the absorption range was between 300-800 nm [52]. This confirms the formation of iron oxide nanoparticles. From the appearance of a broadband and the absence of any hump spectra, it may be concluded that not much size difference was present in the synthesized nanoparticles.

UV-Visible Spectroscopy
The UV-Vis spectra ( Figure 2) exhibited the characteristic continuous peak absorption of both the magnetite nanoparticles in the visible range; the absorption range was between 300-800 nm [52]. This confirms the formation of iron oxide nanoparticles. From the appearance of a broadband and the absence of any hump spectra, it may be concluded that not much size difference was present in the synthesized nanoparticles.

FTIR Analysis of JC-Fe3O4 Nanoparticles
Next the FTIR analysis was performed to prove the presence of JC latex as the capping material for the synthesized MNPs. Synthesized JC-Fe3O4 NPs showed a strong absorption band at 1607 cm −1 (stretching vibration of C-N group), which was attributed to the binding of JC latex as the capping agent since this peak was also observed to be a significant peak in case of dried JC latex powder at 1618 cm −1 [37]. Other significant FTIR peaks showed the bands at 3248 cm −1 (N-H stretching for amides), 2923 cm −1 (secondary amine), 1373 cm −1 (-CO-stretching), and 1070 cm −1 (O-H stretching), which clearly proved the presence of protein/peptide on the nanoparticle binding surface. This data also fitted well with the previously reported Jatropha curcas extract capped nanoparticles [53] (Figure

FTIR Analysis of JC-Fe 3 O 4 Nanoparticles
Next the FTIR analysis was performed to prove the presence of JC latex as the capping material for the synthesized MNPs. Synthesized JC-Fe 3 O 4 NPs showed a strong absorption band at 1607 cm −1 (stretching vibration of C-N group), which was attributed to the binding of JC latex as the capping agent since this peak was also observed to be a significant peak in case of dried JC latex powder at 1618 cm −1 [37]. Other significant FTIR peaks showed the bands at 3248 cm −1 (N-H stretching for amides), 2923 cm −1 (secondary amine), 1373 cm −1 (-CO-stretching), and 1070 cm −1 (O-H stretching), which clearly proved the presence of protein/peptide on the nanoparticle binding surface. This data also fitted well with the previously reported Jatropha curcas extract capped nanoparticles [53] (Figure 3a).

FTIR Analysis of CT-Fe 3 O 4 Nanoparticles
Similarly, the FTIR peak analysis of CT-Fe 3 O 4 NPs revealed that a broadband 3280 cm −1 was due to O-H stretching from the eugenol-OH present in the aqueous extract of CT leaf. The other significant bands at 1620 cm −1 (for carbonyl stretching) match well with the reported CT extract IR at 1638 cm −1 [54]; 2922 cm −1 (for C-H stretching) and 1059 cm −1 (for C-O stretching vibration) confirmed the formation of CT leaves extract-coated MNPs [55] (Figure 3b). Similarly, the FTIR peak analysis of CT-Fe3O4 NPs revealed that a broadband 3280 cm −1 was due to O-H stretching from the eugenol-OH present in the aqueous extract of CT leaf. The other significant bands at 1620 cm −1 (for carbonyl stretching) match well with the reported CT extract IR at 1638 cm −1 [54]; 2922 cm −1 (for C-H stretching) and 1059 cm −1 (for C-O stretching vibration) confirmed the formation of CT leaves extract-coated MNPs [55] (Figure 3b).

FE-SEM Analysis
Analysis of FE-SEM images for JC-Fe3O4 and CT-Fe3O4 NPs showed the surface morphology of respective NPs was round-shaped. The size ranges for JC-Fe3O4 and CT-Fe3O4 were 20-42 nm ( Figure   40 60 80   Similarly, the FTIR peak analysis of CT-Fe3O4 NPs revealed that a broadband 3280 cm −1 was due to O-H stretching from the eugenol-OH present in the aqueous extract of CT leaf. The other significant bands at 1620 cm −1 (for carbonyl stretching) match well with the reported CT extract IR at 1638 cm −1 [54]; 2922 cm −1 (for C-H stretching) and 1059 cm −1 (for C-O stretching vibration) confirmed the formation of CT leaves extract-coated MNPs [55] (Figure 3b).

FE-SEM Analysis
Analysis of FE-SEM images for JC-Fe3O4 and CT-Fe3O4 NPs showed the surface morphology of respective NPs was round-shaped. The size ranges for JC-Fe3O4 and CT-Fe3O4 were 20-42 nm ( Figure  5a) and 26-35 nm (Figure 5b), respectively, and both were well surrounded by the respective green 40 60 80    (Figure 5b), respectively, and both were well surrounded by the respective green coating. The images confirmed that the formation of natural product-based nanoparticles had a spherical shape. coating. The images confirmed that the formation of natural product-based nanoparticles had a spherical shape.

Dye Adsorption Study
The adsorption of MB as a model pollutant was performed to evaluate the adsorption ability of synthesized JC-Fe3O4 and CT-Fe3O4 NPs. Two model equilibrium adsorption isotherms, viz., Langmuir adsorption isotherm and Freundlich adsorption isotherm, were applied. For better understanding of the adsorption process, it is important to investigate the relevant kinetics; two different common kinetic models, pseudo-first-order and pseudo-second-order models were studied.

Dye Adsorption Isotherm of MB Dye
The Langmuir isotherm accounts for the monolayer surface coverage of the adsorbents while the Freundlich isotherm defines for multilayer adsorption. The linear form of Langmuir and Freundlich isotherm equations are depicted as: Langmuir model: Freundlich model: log Qe = log KF + (1/n) log Ce The adsorption capacity is calculated by the equation: Another adsorption parameter, RL, correlation factor at equilibrium, is also calculated for adsorption of MB on for Langmuir isotherm using the equation: For 0 < RL < 1, adsorption process is satisfactory, and for RL ≥ 1 it is unfavourable. Where, Co = initial concentration of adsorbate in mg/L, Ce = equilibrium concentration of

Dye Adsorption Study
The adsorption of MB as a model pollutant was performed to evaluate the adsorption ability of synthesized JC-Fe 3 O 4 and CT-Fe 3 O 4 NPs. Two model equilibrium adsorption isotherms, viz., Langmuir adsorption isotherm and Freundlich adsorption isotherm, were applied. For better understanding of the adsorption process, it is important to investigate the relevant kinetics; two different common kinetic models, pseudo-first-order and pseudo-second-order models were studied.

Dye Adsorption Isotherm of MB Dye
The Langmuir isotherm accounts for the monolayer surface coverage of the adsorbents while the Freundlich isotherm defines for multilayer adsorption. The linear form of Langmuir and Freundlich isotherm equations are depicted as: Freundlich model: log Q e = log K F + (1/n) log C e The adsorption capacity is calculated by the equation: Another adsorption parameter, R L , correlation factor at equilibrium, is also calculated for adsorption of MB on for Langmuir isotherm using the equation: For 0 < R L < 1, adsorption process is satisfactory, and for R L ≥ 1 it is unfavourable. Where, C o = initial concentration of adsorbate in mg/L, C e = equilibrium concentration of (adsorbate + adsorbent), Q e = adsorption capacity in mg/g, K L = Langmuir constant, Q m = maximum adsorption capacity, K F = Freundlich constant, n = separation factor, V = total volume of the solution in L, m = amount of adsorbent in g.
It was observed from Figure 6a

Toxic Metal Adsorption Study (with Concentration)
There are reports in the existing literature that MNPs have the potential to adsorb heavy as well as toxic metal ions such as Hg 2+ ,Cd 2+ ,Pb 2+ ,Co 2+ ,Cu 2+ etc [43,[58][59][60]. We have investigated the ability to remove toxic metal ions Co 2+ and Cu 2+ from aqueous medium via adsorption onto the synthesized JC-Fe3O4 and CT-Fe3O4 NPs. To understand the nature of adsorption of the metal ions, we applied our experimental data to the Langmuir and Freundlich adsorption isotherms; the results are presented in Figure 7a (for Co 2+ ), Figure 7b (for Cu 2+ ), and in Table 3. Table 4 represents a compilation of literature value of adsorption capacity of different adsorbent for Co 2+ and Cu 2+ . From Table 3, it is  Table 1 supports that for MB dye adsorption, Freundlich model fits better than Langmuir model in case of JC-Fe 3 O 4 NPs as the values of R 2 in the case of Freundlich model are much closer to 1. NPs wasinvestigated using pseudo-first-order and pseudo-second-order kinetic models. Pseudo-first order and pseudo-second order kinetics were investigated by the following equations [57]: Pseudo-second-order: t/Q t = 1/K 2 Q e 2 + t/Q e (8) Q t was calculated by the equation: where, K 1 = rate constant of first-order kinetics in min −1 , K 2 = rate constant of second order kinetics in g/mg·min, Q e = adsorption capacity at equilibrium in mg/g, and Q t = adsorption capacity in mg/g at time t. All the experimental and calculated data based on the above kinetic models for adsorption of MB on JC-Fe 3 O 4 NPs are presented in Table 2. From the results in Table 2 and Figure 6d, it is seen that although the correlation coefficient (R 2 ) is almost similar for both the kinetic models, the experimental value of the adsorption capacity Q e (expt) agrees better with the calculated value Qe (cal) based on pseudo-second-order model than with pseudo-first-order model, indicating that our adsorption process follows the former model. No consistent results were observed for the dye adsorption on CT-Fe 3 O 4 NPs.

Toxic Metal Adsorption Study (with Concentration)
There are reports in the existing literature that MNPs have the potential to adsorb heavy as well as toxic metal ions such as Hg 2+ ,Cd 2+ ,Pb 2+ ,Co 2+ ,Cu 2+ , etc. [43,[58][59][60]. We have investigated the ability to remove toxic metal ions Co 2+ and Cu 2+ from aqueous medium via adsorption onto the synthesized JC-Fe 3 O 4 and CT-Fe 3 O 4 NPs. To understand the nature of adsorption of the metal ions, we applied our experimental data to the Langmuir and Freundlich adsorption isotherms; the results are presented in Figure 7a (for Co 2+ ), Figure 7b (for Cu 2+ ), and in Table 3. Table 4 represents a compilation of literature value of adsorption capacity of different adsorbent for Co 2+ and Cu 2+ . From Table 3, it is evident that the values of the correlation coefficient (R 2 ) for all the ions are greater for Langmuir plot than for Freundlich case, implying that the metal ion adsorption process follows the Langmuir model better than the Freundlich model. The observed maximum adsorption capacity (Qm) of CT-Fe 3 O 4 NPs is 513.7 and 463.23 mg/g for Co 2+ and Cu 2+ respectively. The corresponding values are 501.3 and 543.3 mg/g for JC-Fe 3 O 4 . These values are much better than all other reported values, as seen in Table 4.

Magnetic Properties
In order to show the magnetic behavior of JC-Fe3O4 and CT-Fe3O4 NPs, the dispersed solutions ( Figure 8a) were treated with magnet externally, and the nanoparticles were found to get deposited near the magnet (Figure 8b). This observation also exhibited the possibility of using the powerful magnetic field for the separation of MNPs after wastewater treatment.

Magnetic Properties
In order to show the magnetic behavior of JC-Fe 3 O 4 and CT-Fe 3 O 4 NPs, the dispersed solutions ( Figure 8a) were treated with magnet externally, and the nanoparticles were found to get deposited near the magnet (Figure 8b). This observation also exhibited the possibility of using the powerful magnetic field for the separation of MNPs after wastewater treatment.

Magnetic Properties
In order to show the magnetic behavior of JC-Fe3O4 and CT-Fe3O4 NPs, the dispersed solutions ( Figure 8a) were treated with magnet externally, and the nanoparticles were found to get deposited near the magnet (Figure 8b). This observation also exhibited the possibility of using the powerful magnetic field for the separation of MNPs after wastewater treatment. Magnetic properties of the synthesized JC-Fe3O4 and CT-Fe3O4 NPs were also studied with the help of VSM (Vibrating sample magnetometer). Figure 8c,d gives the changes in the magnetization with the applied magnetic field. The superparamagnetic natures of the nanoparticles were confirmed by the absence of the hysteresis loop. The saturation magnetization for JC-Fe3O4 and CT-Fe3O4 NPs was found to be 38.46 and 34.35 emu/g, respectively.

Characterization of Bacteria Isolated from Pond Water
The collected pond water (without nanoparticles) was serially diluted, a single colony was isolated, and pure culture was generated. Gram staining was performed on the isolated bacterial culture and was found to be Gram-positive bacteria.

Wastewater Treatment
Fe3O4 NPs had been previously reported in the literature to exhibit antibacterial activity [69][70][71]. Similarly, CT leaves were also reported to show antibacterial efficacy [35] as well as JC latex [53,72]. Hence, the antibacterial activity of CT-Fe3O4 and JC-Fe3O4 NPs were examined. The amount of bacteria colony was observed to be reduced by more than 50% in the case of the pond water treated with CT-Fe3O4 (Figure 9b) and JC-Fe3O4 NPs (Figure 9c) compared to the pond water that was not treated (Figure 9a) with any of the antibacterial agents. The CFU value also showed that the amount of bacteria colony of water treated with nanoparticles was being reduced with increasing the amount of nanoparticles. These observations confirmed the antibacterial activity of CT-Fe3O4 and JC-Fe3O4 against various types of water-borne bacteria (Figure 9d).

Characterization of Bacteria Isolated from Pond Water
The collected pond water (without nanoparticles) was serially diluted, a single colony was isolated, and pure culture was generated. Gram staining was performed on the isolated bacterial culture and was found to be Gram-positive bacteria.

Wastewater Treatment
Fe 3 O 4 NPs had been previously reported in the literature to exhibit antibacterial activity [69][70][71]. Similarly, CT leaves were also reported to show antibacterial efficacy [35] as well as JC latex [53,72]. Hence, the antibacterial activity of CT-Fe 3 O 4 and JC-Fe 3 O 4 NPs were examined. The amount of bacteria colony was observed to be reduced by more than 50% in the case of the pond water treated with CT-Fe 3 O 4 ( Figure 9b) and JC-Fe 3 O 4 NPs (Figure 9c) compared to the pond water that was not treated (Figure 9a) with any of the antibacterial agents. The CFU value also showed that the amount of bacteria colony of water treated with nanoparticles was being reduced with increasing the amount of nanoparticles. These observations confirmed the antibacterial activity of CT-Fe 3 O 4 and JC-Fe 3 O 4 against various types of water-borne bacteria (Figure 9d).
Hence, the antibacterial activity of CT-Fe3O4 and JC-Fe3O4 NPs were examined. The amount of bacteria colony was observed to be reduced by more than 50% in the case of the pond water treated with CT-Fe3O4 ( Figure 9b) and JC-Fe3O4 NPs (Figure 9c) compared to the pond water that was not treated (Figure 9a) with any of the antibacterial agents. The CFU value also showed that the amount of bacteria colony of water treated with nanoparticles was being reduced with increasing the amount of nanoparticles. These observations confirmed the antibacterial activity of CT-Fe3O4 and JC-Fe3O4 against various types of water-borne bacteria (Figure 9d (Figure 10a,b). In the case of E. coli, ZOI was found to be the same (i.e., 7 mm) for both MNPs (Figure 10c,d). However, against S. aureus for CT-Fe 3 O 4 the ZOI was 8 mm, whereas for CT-Fe 3 O 4 it was 6.5 mm (Figure 10e,f). Based on the above result, it was observed that both CT-Fe 3 O 4 and JC-Fe 3 O 4 NPs exhibited quite effective antibacterial property against both Gram-positive and Gram-negative bacteria. The relative antibacterial activity of the two synthesized nanoparticles has been summarized in Figure 10g.

Disk Diffusion
The antibacterial activities of CT-Fe3O4 and JC-Fe3O4 NPs measured in terms of zone of inhibition (ZOI) are shown in Figure 10. It was observed that ZOI against water-born Gram-positive bacteria for CT-Fe3O4 and JC-Fe3O4 NPs showed a diameter of 10 mm and 7 mm respectively (Figure 10a,b). In the case of E.coli, ZOI was found to be the same (i.e., 7 mm) for both MNPs (Figure 10c,d). However, against S.aureus for CT-Fe3O4 the ZOI was 8 mm, whereas for CT-Fe3O4 it was 6.5 mm (Figure 10e,f). Based on the above result, it was observed that both CT-Fe3O4 and JC-Fe3O4 NPs exhibited quite effective antibacterial property against both Gram-positive and Gram-negative bacteria. The relative antibacterial activity of the two synthesized nanoparticles has been summarized in Figure 10g.  Gram-positive bacteria, for CT-Fe3O4, the MIC was 250 ppm and for JC-Fe3O4 it was 500 ppm ( Figure  11). From the MIC, we got the expected results similar to disk diffusion test i.e., both CT-Fe3O4 and JC-Fe3O4 NPs, which showed decent antibacterial property. CT-Fe3O4 NPs were found to be more effective than JC-Fe3O4 NPs against both the Gram-positive and Gram-negative bacteria.

DPPH Scavenging Assay
The antioxidant properties of both the nanoparticles (JC-Fe3O4 and CT-Fe3O4NPs) are shown in Figure 12. The DPPH scavenging assay of the respective MNPs resulted in IC50 values of 0.30 mg/mL for JC-Fe3O4, and for CT-Fe3O4 it was 0.67 mg/mL; and the IC50 value for the uncoated Fe3O4 nanoparticle was estimated to be 1.40 mg/mL. The chosen standard (positive control) was the gallic acid solution in methanol. All the concentrations were taken as 0.06 mg/mL, 0.25 mg/mL, 0.57 mg/mL, 1.00 mg/mL, and 1.57 mg/mL in methanol. Gram-positive bacteria, for CT-Fe3O4, the MIC was 250 ppm and for JC-Fe3O4 it was 500 ppm ( Figure  11). From the MIC, we got the expected results similar to disk diffusion test i.e., both CT-Fe3O4 and JC-Fe3O4 NPs, which showed decent antibacterial property. CT-Fe3O4 NPs were found to be more effective than JC-Fe3O4 NPs against both the Gram-positive and Gram-negative bacteria.

DPPH Scavenging Assay
The antioxidant properties of both the nanoparticles (JC-Fe3O4 and CT-Fe3O4NPs) are shown in Figure 12. The DPPH scavenging assay of the respective MNPs resulted in IC50 values of 0.30 mg/mL for JC-Fe3O4, and for CT-Fe3O4 it was 0.67 mg/mL; and the IC50 value for the uncoated Fe3O4 nanoparticle was estimated to be 1.40 mg/mL. The chosen standard (positive control) was the gallic acid solution in methanol. All the concentrations were taken as 0.06 mg/mL, 0.25 mg/mL, 0.57 mg/mL, 1.00 mg/mL, and 1.57 mg/mL in methanol.

Measurement of Cytotoxicity Using MTT Assay
In addition to more efficient water purifying capabilities of the natural product-coated MNPs, it is important to study the overall toxicity associated with them. Since treated water, may consist of

Measurement of Cytotoxicity Using MTT Assay
In addition to more efficient water purifying capabilities of the natural product-coated MNPs, it is important to study the overall toxicity associated with them. Since treated water, may consist of residual MNPs in ppm level due to inefficient removal process. It is reported that coated MNPs also showed lower cytotoxicity towards cancerous cells than the uncoated one [73]. Hence, to investigate whether the synthesized coated MNPs are toxic to human cells, cytotoxicity was investigated against human cancer cell lines (SW480 and HeLa) by MTT assay. Each cell line was incubated with both the MNPs for 48 h in different concentrations (Figure 13), and then the percentage viability of the cells was estimated. The percentage viability of the cells was found to be little enhanced in CT-Fe 3 O 4 and almost remained the same in JC-Fe 3 O 4 . Based on the in vitro cytotoxicity results, it can be concluded that the MNPs did not exhibit cytotoxicity towards both cell lines, indicating these MNPs are not harmful to human cells. These results were well corroborated with the previous literature with natural product-coated MNPs [74].

Conclusions
The present study reports the green syntheses of two natural products, coated JC-Fe3O4 and CT-Fe3O4 NPs. Both the synthesized MNPs are effective in removing the content of wastewater like organic dyes and toxic metal ions. The study also shows that the nanoparticles are effective as antibacterial agents (both Gram-positive and Gram-negative bacteria) as well as antioxidant agents. Both the coated MNPs also do not exhibit any cytotoxic effect, as shown by the MTT assay. Therefore, JC-Fe3O4 and CT-Fe3O4 NPs both show promise for environment-friendly composites for effective water treatment.