Photoremediation of methylene blue by biosynthesized ZnO/Fe 3 O 4 nanocomposites using Callistemon viminalis leaves aqueous extract: A comparative study

: This article reports a simple, cost - e ﬀ ective, and eco - friendly biosynthesis of ZnO/Fe 3 O 4 nanocomposites using Callistemon viminalis leaves ’ water extract. For the ﬁ rst time, we used a green synthetic route via C. viminalis leaves ’ extract to prepare ZnO/Fe 3 O 4 nanocomposites ( NCs ) using zinc acetate and ferric chloride as precursor materials. Fourier transform infrared ( FTIR ) spectroscopic results revealed polyphenolic compounds mainly phenolic acids present in the plant extract acted as both reducing and stabilizing agents to synthesize ZnO/Fe 3 O 4 NCs. ﬀ extract ) nanoparticles were exam ined for photodegradation of methylene blue under visible light irradiation for 150 min. The result reveals that the photodegradation e ﬃ ciency of ZnO/Fe 3 O 4 NCs ( 99.09% ) was higher compared to that of monometallic ZnO ( 84.7% ) and Fe 3 O 4 ( 37.1% ) nanoparticles.


Introduction
Industrialization and urbanization have increased water pollution to a great extent because of the direct disposal of organic and industrial waste into water bodies [1]. Among all, dying industries produce an enormous amount of wastewater containing unused dye along with other chemicals [2]. It reported that the majority of dyes are toxic and nonbiodegradable [3]. Hence, the dye effluent contaminates surface and underground water, which brings out adverse effects on flora and fauna [4,5]. Methylene blue (MB) is the most popular thiazine dye used in textile industries. The prolonged exposure to MB results in harmful effects, such as cyanosis, skin irritation, and gastrointestinal irritation, in living beings [6]. To solve this challenge, many physical and chemical techniques including flocculation-coagulation, surface adsorption, ion-exchange, chemical precipitation, and photocatalytic degradation have been used to remove the dye from waste water [7,8].
Because of the simple experimental procedure and decomposition of organic dye molecules into nontoxic simple  products in the presence of semiconductors under proper light irradiation, the photodegradation process proves its superiority over other methods [9,10]. For semiconductorassisted photocatalytic process, various materials, such as ZnO, CuO, TiO 2 , and more, have been widely used as photocatalysts previously [11][12][13]. Among different semiconductor materials, ZnO is a nontoxic, easily available, and costeffective material with a bandgap of 3.2 and 60 MeV exciton binding energy [14]. That is why ZnO became the priority to many researchers working in the photocatalytic degradation of dyes using semiconductors. But ZnO semiconductor has a rapid tendency of recombination of photo-induced electron-hole pairs, which makes it difficult to gain the practical demand [15]. To overcome this problem, many metallic or nonmetallic materials, such as CdS, Ag 2 O, CuO, and g-C 3 N 4 , have doped with ZnO and showed improved efficiency toward photodegradation process [16][17][18][19].
However, the removal of nanocomposites (NCs) from the treated solution is very tedious and expensive, which is still a challenge. In this concern, magnetite nanoparticles (Fe 3 O 4 NPs) play an effective role as they are easily separable from the solution by applying an external magnetic field [20]. Apart from this, doping of metallic NPs with magnetite NPs improves their functionality and recyclability and in turn cost effectiveness of the process [21,22]. Inspired by this, many researchers have synthesized and investigated the results of magnetite NCs [23]. After reviewing the literature, we observed that various approaches, such as sol-gel, hydrothermal synthesis, precipitation, and microemulsion, have been adopted for preparing magnetite composites [24][25][26][27]. But many of these methods have certain demerits, such as the use of expensive and hazardous chemicals and the formation of toxic byproducts, which makes it difficult to achieve the requirement of green synthesis. In recent decades, the use of bioproducts (including biomolecules, bacteria, fungi, or plant extracts) for the synthesis of NPs has attracted researchers as well [28]. Metals and metal oxide nanoparticles have also been used as homogeneous or heterogeneous nanocatalysts in various organic syntheses due to the large surface-to-volume ratio of nanoparticles compared to bulk materials [29,30]. This strategy provides a simple, cost-effective, and eco-friendly route for the fabrication of nanomaterials [31]. Although synthesis of ZnO/Fe 3 O 4 NCs by different routes has been reported [32,33], a few reports on a green synthesis of ZnO/Fe 3 O 4 NCs are available [34,35]. In our present study, we have synthesized ZnO/Fe 3 O 4 NCs using Callistemon viminalis leaves' extract as a green reducing and stabilizing agent. C. viminalis is a small tree that belongs to the family Myrtaceae with a characteristic brushlike flowers. It is a traditional medicine to treat hemorrhoids, gastroenteritis, diarrhea, and skin infection [36][37][38]. The phytochemical study reported that C. viminalis is rich in biomolecules, including viminadiones, quercetin, and betulinic acid, and can act as a green reducing and capping agent during the fabrication of nanomaterials.
After reviewing the literature, we understand that C. viminalis leaves' extract-mediated biomimetic synthesis of ZnO/Fe 3 O 4 has not been reported till date. In this study, we have attempted to understand the role of biomolecules present in leaves' extract as a reducing and stabilizing agent for the fabrication of ZnO/Fe 3

Preparation of C. viminalis leaves' extract
Leaves of C. viminalis were collected from Jaipur National University campus, India. The collected leaves were thoroughly washed under tap water and finally washed using deionised water. After drying in shade, the leaves were powdered in an electrical grinder. About 25 g of the powdered leaves in 100 mL deionised water was refluxed in a Soxhlet apparatus (Sigma-Aldrich, India) for 2 h at 80°C on a magnetic stirrer. On cooling, the suspension was filtered through Whatmann's filter paper, and the filtrate was collected as leaves' extract and stored in a refrigerator at 2°C for further studies.

Biosynthesis of ZnO/Fe 3 O 4 NCs
The ZnO/Fe 3 O 4 NCs were prepared through an eco-friendly green route, and the synthesis procedure is briefly illustrated as follows: 30 mL of C. viminalis leaves' extract was added slowly in a round-bottom flask containing 30 mL of zinc acetate solution (0.01 M) under stirring. After 10 min, 0.16 g of ferric chloride in 10 mL deionised water was introduced dropwise into the flask and heated to 60°C, followed by the addition of NaOH (0.1 M) to maintain pH 10. The color of the solution changes from black to blackish brown after 1 h stirring, which indicated the formation of ZnO/Fe 3 O 4 NCs. Afterward, the solution was cooled to room temperature, centrifuged, collected in a China dish, washed several times with ethanol to remove unused extract and NaOH, dried in an oven at 80°C, and finally calcined at 300°C before storing for further studies. For comparison, monometallic nanoparticles (ZnO and Fe 3 O 4 ) had also been synthesized using C. viminalis leaves' extract. A schematic of aforementioned green synthesis is shown in Figure 1.

Characterization
FTIR spectral analysis in the range of 4,000-400 cm −1 was carried out to investigate the role of C. viminalis leaf extract in the fabrication of ZnO, Fe 3 O 4 , and ZnO/Fe 3 O 4 nanoproducts using a PerkinElmer spectrophotometer (MNIT, Jaipur). A powder X-ray diffraction technique was performed to determine the crystallinity and particle size of biosynthesized product by PAN analytical (XPART PRO) diffractometer in the scattering range (2θ) of 20-80°using Cu Kα radiation (λ = 1.5406 Å). The surface morphology of green synthesized samples was determined by scanning electron microscopy (SEM) using Nova Nano SEM 450 (MNIT, Jaipur) and transmission electron microscopy (TEM) at IIT Roorkee. The elemental composition was investigated using EDX analysis. Chemical states of elements present in ZnO/Fe 3 O 4 NCs were analyzed by X-ray photoelectron spectroscopic technique (XPS, PHI 5000 Versa Probe III, IIT Roorkee). The thermal stability of ZnO/Fe 3 O 4 sample was recorded in a nitrogen atmosphere at a heating rate of 5°C min −1 by TGA/DTA analyzer (EXSTAR TG/DTA 6300, IIT Roorkee).

Designing of the photocatalytic activity experiment
The photocatalytic efficiency of biosynthesized ZnO, Fe 3 O 4 , and ZnO/Fe 3 O 4 nanomaterials for the degradation of MB dye was evaluated under visible light at pH 7. For the photodegradation study, three experimental sets were prepared. Each set comprised seven beakers (100 mL) with 25 mL solution of MB (32 mg L −1 ) in each. The dose of biosynthesized ZnO, Fe 3 O 4 , or ZnO/Fe 3 O 4 nanoproducts taken was 0.004 g in each beaker. After certain intervals of time (15,30,45,60,75,90, and 150 min), one beaker from each set was removed from irradiation, and dye solutions were centrifuged at 8,000 rpm followed by filtration to remove the photocatalyst. MB degradation was examined by measuring the absorbance of the dye solution at λ max = 665 nm using a UV-Vis spectrophotometer. The percentage of MB degradation was determined by the following equation: In equation (1), η is the degradation percentage and A 0 and A t are the absorbances of MB dye solution at t = 0 and after time t, respectively.
3 Results and discussion

FTIR analysis
The involvement of biomolecules present in C. viminalis leaves' extract, for the fabrication of nanomaterials, was screened by FTIR spectroscopic analysis. Figure 2  were assigned to O-H stretching of phenolic acids and phenols, C-H stretching in CH 3 and CH 2 , C]O groups in phenolic acids and flavonoids, C]C stretching of the aromatic ring, and C-H deformation in CH 3 and C-OH stretching in phenolic acids, respectively, as reported in various literature [39][40][41]. However, after the reduction of metal precursors into their respective metal nanoparticles, a remarkable difference in intensity, position, and shape of absorption peaks had been observed, which showed the participation of biomolecules (present in leaves' extract) in the reduction and capping of nanomaterials. The additional peaks in the spectra of monometallic NPs at 466.53 cm −1 (Figure 2  suggests that polyphenolic compounds mainly phenolic acids are responsible for the bioreduction of metal ions and capping of as-prepared nanoproducts.

Mechanism of biosynthesis
On the basis of FTIR results, a possible mechanism for the C. viminalis leaves' extract-mediated synthesis of ZnO, Fe 3 O 4 , and ZnO/Fe 3 O 4 has been proposed.
In short, betulinic acid present in leaves' extract undergoes oxidation according to the free radical mechanism, that is, betulinic acid to dehydro betulinic acid (Scheme 1).
Zn +2 /Fe +3 ions (present in solution) form complex with dehydro betulinic acid via transfer of electrons from anionic dehydro betulinic acid to metal ions. On calcination, the resulting complex is converted into respective metal oxide nanoparticles because of the capping effect of biomolecules [43].

XRD analysis
The phase and crystal structure of biosynthesized ZnO, Fe 3 O 4 , and ZnO/Fe 3 O 4 nanomaterials were examined by powder X-ray diffraction analysis. Figure 3 depicts the

SEM and TEM analysis
Morphology and nanoscale of the biosynthesized samples were analyzed using SEM and TEM. An overview of SEM images of Zn, Fe 3 O 4 , and ZnO/Fe 3 O 4 samples is shown in Figure 4(a-c) Morphological differences among as-prepared samples were also identified by TEM micrographs (Figure 4(d-f)). The spherical shape of ZnO NPs with a particle size of ∼45 nm can be seen in Figure 4

EDX analysis
To determine the chemical composition of ZnO, Fe 3 O 4 , and ZnO/Fe 3 O 4 nanoproducts, EDX analysis was carried out and results are displayed in Figure 5(a-c). It can be seen in Figure 5(a) that the EDX spectrum consists of strong peaks for Zn and O, whereas Figure 5(b) shows Fe and O elemental peaks. In case of ZnO/Fe 3 O 4 NCs ( Figure 5(c)), strong signals for Zn, Fe, and O elements were well recognized, which further confirmed the coexistence of ZnO and Fe 3 O 4 . The appearance of carbon in all three spectra may be due to biomolecular capping on the surface of nanoproducts [45]. Based on EDX outcomes, the weight percentage of elements in ZnO/Fe 3 O 4 NCs was 32.56, 38.67, and 28.77% for Zn, Fe, and O, respectively.

XPS analysis
XPS analysis was carried out to demonstrate the chemical nature of the surface of biosynthesized ZnO/Fe 3 O 4 NCs.  Figure 6(b-e). In Figure 6(b), a doublet for Fe2p at 711.94 and 726.93 eV was assigned to respective binding energies of Fe2p 3/2 and Fe2p 1/2 of Fe 3 O 4 [46,47]. In the Zn2p spectra (Figure 6(c)), the spin-orbit doublet Zn2p 3/2 and Zn2p 1/2 peaks were centered at binding energies of 1021.7 and 1044.65 eV, respectively [48]. It is evident from the literature that Zn2p 3/2 and Zn2p 1/2 peaks are separated by 23 eV in pure ZnO. From the XPS results, peaks of Zn2p in NCs were separated by 21 eV, which strongly manifests the synergistic effect between ZnO and Fe 3 O 4 NPs, contributing to the enhancement in photocatalytic activity of NCs [49]. XPS spectrum of C1s (Figure 6(d)), consists of three peaks at 283 eV (C-C) [50], 284 eV (C]C) [46,51], and 285 eV (C]O) [52], attributed to the polyphenolic compounds of leaves' extract acting as a stabilizing agent for ZnO/Fe 3 O 4 NCs [53]. In the O1s spectrum shown in

Thermal analysis
Thermal characteristics of ZnO/Fe 3 O 4 NCs were determined simultaneously in a single run by using TGA and DTA (Figure 7). TGA results showed that the thermal decomposition of ZnO/Fe 3 O 4 NCs occurred in four steps. Initially, up to 100°C, the weight loss of 5.2% was due to the loss of adsorbed water on the surface of NCs. The weight loss observed in the second step (200-400°C) was 17.3%, which might be due to the dismissal of biomolecules capped on the surface of NCs. The loss in weight observed in between 400 and 600°C (third step) was assigned to the adsorbed oxygen species [56]. In the last step, a loss of 7.5% in weight was observed up to 800°C. DTA thermogram (Figure 7) displayed energy changes irrespective of change in weight. The peaks observed at 328 and 599°C were associated with the release of bioactive molecules and adsorbed oxygen,

Assessment of the photocatalytic activity
The photodegradation of MB in the presence of as-prepared nanoproducts was examined under visible light, and the extent of degradation was measured in terms of absorbance of MB solution using a UV-Vis spectrophotometer after certain intervals of time for 150 min. The results of degradation studies are showcased in Figures 8 and 9, and Table 1, which revealed that the degradation of MB increases with an increasing irradiation time. From the absorbance spectra of MB ( Figure 8 and Moreover, the degradation efficiency of ZnO/Fe 3 O 4 NCs was examined for three consecutive runs, and the results are shown in Figure 10. From the results, it is clear that the composites were active up to three cycles. Although in the third cycle, the degradation efficiency was decreased (90.1%). This decrement may be due to the adsorbance of some MB on the surface of photocatalyst, which perhaps blocks some active sites of NCs and can also be due to some loss of NCs during the recovery process.
The possible mechanism for the photodegradation of MB dye over biosynthesized ZnO/Fe 3 O 4 NCs under visible light is shown in Figure 11.
The phenomenon of photodegradation of MB takes place when visible light is irradiated on the photocatalyst (ZnO, Fe 3 O 4 , or ZnO/Fe 3 O 4 ), which leads to the generation of electron-hole pairs in conduction/valence bands (VBs) simultaneously on the surface of the photocatalyst  (equation (2)). In the conduction band (CB), oxygen on the surface of the photocatalyst combines with the excited electron and forms˙− O 2 (superoxide radical; equation (3)). This radical checks the recombination of e − /h + pairs by converting into˙OH radicals through hydroperoxyl radicals (HOO˙) and H 2 O 2 intermediates (equation (4)). Simultaneously, holes produced in VB react with the surface water to produce hydroxyl radicals (˙OH; equation (5)). The hydroxyl radicals produced in CB and VB, on the surface of photocatalyst, act as a strong oxidizing agent, which in turn degrade MB molecules into simple inorganic molecules, such as water, carbon dioxide, and inorganic ions (equation (6)). The survey of the previous literature also reveals that˙− O 2 (superoxide) radicals and hydroxyl radicals (˙OH) are the leading reactive species for the photodegradation of MB dye [57].
As shown in Figure 8 and Table 1, the absorption intensity of dye gradually decreases with an increasing irradiation time and finally diminished in case of ZnO/Fe 3 O 4 . This is because of a breakdown of the heterocyclic conjugated structure of MB molecule into simple molecules, such as water, carbon dioxide, and inorganic ions. Apart from this, the photocatalytic efficiency of ZnO/Fe 3 O 4 NCs was greater compared to ZnO NPs, and this can be summarized as follows: Fe 3 O 4 NPs possess a narrow bandgap, and hence, e − /h + pairs produced in it under irradiation recombines fastly, as a result charge carriers could not survive for a long time for the photocatalysis process [58]. In ZnO/Fe 3 O 4 NCs, the energy level of CB and VB of ZnO differs from that of Fe 3 O 4 . During irradiation, some photogenerated electrons from ZnO are captured by Fe 3 O 4 NPs at the composite's interface, where they react with the surface oxygen to form superoxide radicals. However, some holes transfer to VB of Fe 3 O 4 from ZnO and react with the surface water to produce hydroxyl radicals (˙OH). This phenomenon at the interface restricts an electron-hole recombination in ZnO. As a resultant, the generation of reactive species increases at the junction of ZnO-Fe 3 O 4 , which accelerates the degradation of dye molecules [59]. Hence, the efficient charge transfer separation at heterojunction of two different semiconductor materials attributes to an enhanced photo-catalytic activity of ZnO/Fe 3 O 4 NCs to degrade MB