Nanostructured Ag3PO4 from natural phosphate: High-ecient catalytic reduction of 4-nitrophenol and antibacterial studies.

In this paper, a novel approach was successfully developed for preparing nanostructured Ag 3 PO 4 using Moroccan phosphate as a source for phosphorus precursor. The as-synthetized nanomaterial was characterized using various techniques. A pure crystalline Ag 3 PO 4 phase was obtained after drying, exhibiting a mesoporous structure with specic surface area of 35 m 2 g − 1 . The use of this simple, environmentally friendly, and inexpensive procedure can be useful for the development of nanostructured Ag 3 PO 4 catalyst with excellent catalytic activity for reduction of 4-nitrophenol to 4-aminophenol in the presence of NaBH 4 as reducing agent. Furthermore, the Ag 3 PO 4 material was also used as an antibacterial agent against Escherichia-coli and Staphylococcus-aureus bacteria.


Introduction
With industrialization processes and more human activities, the environmental contamination caused by organic pollutants is becoming an overwhelming mystery all over the world [1][2]. The phenolic compounds are considered to be one of the most notorious pollutants generated by industrials sources such as synthetic intermediate in the manufacture of pharmaceuticals, plastics, pigments, dyes, pesticides and fungicidal agents, explosives and industrial solvents [3]. Due to their potential to harm human health and living organisms at low concentrations, these compounds were classi ed as priority materials by the United States Environmental Protection Agency (USEPA) among the top 114 organic pollutants [4][5]. Among the different phenolic compounds, 4-nitrophenol (4-NP) is one of the most frequently occurring products [6]. However, the 4-aminophenol is one of the most important intermediates in the preparing of several analgesic and antipyretic drugs like paracetamol, acetanilide, and phenacetin [7]. Also, it is utilized as corrosion inhibitor in paints, and anticorrosion-lubricating agent in fuels. Further, 4-aminophenol is used e ciently in the dye industry as a wood stain and a dyeing agent for fur and feathers [8]. In view of the harmful effect of 4-NP and the growing demand for 4-AP, the conversion of 4-NP directly to 4-AP via catalytic route becomes greatly desirable. Various metal nanoparticles like as platinum, gold, copper, ruthenium and palladium, are used intensively for the reduction of nitrophenols compounds. All these catalysts are very expensive for industrial use in bulk quantity. To reduce the cost of the catalyst, over the past few years, more attention has been paid to synthesis of silver orthophosphate Ag 3 PO 4 [9,12]. However, low-cost fabrication of well-de ned Ag 3 PO 4 with superior catalytic properties via a simple process remains a great challenge. On the other hand, natural phosphates are an important natural resource in Morocco, which needs to be valorized. They can be employed not only as fertilizers but also, they have been exploited effectively as catalysts in a wide range of organic transformation [13,15]. In continuation of our ongoing program to develop an interesting catalyst at low-cost [16,21], we describe in this paper, a novel chemical wet method-based dissolutionprecipitation reactions using Moroccan natural phosphate (NP) as phosphorus precursor to synthesize of the nanostructured Ag 3 PO 4 as catalyst for the reduction of 4-NP to 4-AP in the presence of sodium borohydride using aqueous phase and its antimicrobial activity against Escherichia coli and Staphylococcus aureus bacteria. To the best of our knowledge, no studies have been performed on the development of Ag 3 PO 4 from natural phosphate and testing its catalytic activity for the reduction of 4-NP to 4-AP, and its antibacterial activity against E. coli and S. aureus in aqueous solution.

The preparation of nanostructured Ag 3 PO 4 catalyst
The Ag 3 PO 4 was prepared by a dissolution/precipitation method from a natural phosphate rock coming from the Khouribga region (Morocco). To use this material prior requires initial treatments such as crushing and washing. The fraction of 200-400μm grain size was washed with distilled water several times to remove the soluble matter. The different elemental constituents of this mineral are given in Table   1. Then, the dissolution process was carried out in a round bottom ask of 500 ml capacity at a rate of 200 rpm. Firstly, about thirty grams of natural phosphate was dissolved in deionized water acidi ed by HNO 3 acid (65%) to pH 2, under continuous stirring at room temperature we obtained H 3 PO 4 and Ca 2+ ions as well as the insoluble matter after solid/liquid separation process by centrifugation. Next, the Ag 3 PO 4 catalyst was prepared by a simple precipitation method. In a typical synthesis, 1.5 grams of AgNO 3 (8.83 mmol) was dissolved in 80 ml deionized water over 10 min, then the ammonia hydroxide solution 28% (0.1M) was added with drop by drop under magnetic stirring to above mixture to form a transparent solution. And then, phosphorus precursor prepared previously from natural phosphate were added gradually to the mixture reaction, the resulting precipitate was magnetically stirred at room temperature for one hour. After that, a yellow precipitate of silver phosphate Ag 3 PO 4 is then obtained by centrifugation and washed several times with deionized water to release any unreacted species such as Ca 2+ and NO 3ions. Finally, the obtained powder Ag 3 PO 4 was dried in desiccator at 80 °C overnight. The schematic illustration of preparation process of nanostructured Ag 3 PO 4 from natural phosphate is exhibited step by step in Scheme 1.
2.3 Characterization of the catalyst X-ray diffraction (XRD) patterns were obtained at room temperature on a Bruker AXS D-8 diffractometer using Cu-Kα radiation in Bragg-Brentano geometry (θ-2θ). Fourier transform infrared (FT-IR) was performed on an ABB Bomem FTLA 2000 spectrometer equipped with a Golden Gate single re ection ATR accessory. Scanning electron microscopy images were recorded on a FEI Quanta 200 microscope after carbon metallization. The TEM micrographs were obtained on a FEI microscope at 120 kV. Speci c surface area was determined from the nitrogen adsorption/desorption isotherms (at-196°C) and measured with a Quantachrome Autosorb-1 automatic analyzer using the BET equation.

Catalytic reduction of 4-nitrophénol to 4-aminophenol
The catalytic activities were evaluated by reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) in a quartz cuvette. In a typical procedure, 100 µL of 4-NP (1mM) and 1 mL NaBH 4 (0.1 M) were placed in a quartz cuvette containing 3 mL of deionised water. After that 5 mg of Ag 3 PO 4 was added into the cuvette to start the reduction reaction. The process of the conversion of 4-NP to 4-AP was followed by UV-Vis spectroscopy at a maximum wavelength of 400nm.

Antibacterial activity of nanostructured Ag 3 PO 4 powder
The antibacterial activity of the nanostructured Ag 3 PO 4 was studied on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) by the standard disk diffusion assay on Muller-Hinton agar medium. All disks and materials were sterilized in an autoclave at 120 °C for 20 min before experiments. The disk diffusion assay was performed by placing a 6 mm disk saturated with 10 µL of Ag 3 PO 4 aqueous dispersions (1000-125µg/mL) onto an agar plate seeded with E. coli or S. aureus. After 24 hours of incubation at 37°C, the diameters of the inhibition zones were measured.

Characterization of nanostructured Ag 3 PO 4
The phase structures of the as-prepared nanostructured Ag 3 PO 4 were investigated by XRD and showed in   To better elucidate the morphology properties of the prepared Ag 3 PO 4 from natural phosphate, SEM analysis was carried out. In the lower magni cation images, the Fig.4a indicates that the surface of nanostructured Ag 3 PO 4 is formed by a large amount of quasi-spheroid particles having hexagonal and cubic structures, while the higher magni cation image, as shown in Fig.4b, clearly reveals the quasispheroid particles through non-uniform diameter polyhedrons. On the other hand, to con rm the chemical composition of the nanostructured Ag 3 PO4, a semi-quantitative elemental analysis was performed, and its EDS spectrum showed in Fig.4c. The obtained results revealed the presence of O, P and Ag elements without any calcium traces indicating that the Ag 3 PO 4 prepared is pure and does not contain any impurities. Note that the presence of carbon and copper peaks is originated from adhesive Cu-carbon tape.
To further demonstrate the porous structure of nanostructured Ag 3 PO 4 , the speci c surface area (S BET ) of the Ag 3 PO 4 powder was calculated from N 2 -sorption measurements and application of the BET method.
As shown in Fig.5a, the sorption isotherm exhibited a type IV isotherm according to the IUPAC classi cation with a distinct hysteresis loop of H3. Its speci c surface area was of 35 m 2 /g and average pore size D p calculated from BJH (Barrett-Joyner-Halenda) method was 3.1 nm and 7.3 nm (Fig.5b).
Comparing with the low values given in the literature, a relatively large porous surface of Ag 3 PO 4 catalyst could provide more active and bene cial sites for the adsorption of target molecules through the active sites of the catalyst, which would promote the catalytic reaction.

Catalytic Reduction of 4-NP to 4-AP
To investigate the catalytic activity of the Ag 3 PO 4 as nanostructured catalyst, the reduction of 4nitrophenol to its corresponding amino derivatives, 4-aminophenol, in the presence of NaBH 4 in aqueous media was chosen as a model reaction (Scheme 2). Currently, the reduction of 4-NP to 4-AP is monitored by UV-vis spectra at their speci c wavelengths 317 nm for 4-NP and 300 nm for 4-AP.
Firstly, the ability of NaBH 4 to reduce 4-NP in absence of our catalyst was examined. As shown in Fig. 6-A, the 4-NP in an aqueous solution has a maximum absorption at 317. After added NaBH 4 into solution, the absorbance peak of 4-NP was red shifted from 317 to 400 nm immediately along with a colour change from light yellow to bright yellow. This peak was due to the formation of 4-nitrophenolate ions in alkaline condition caused by the addition of reducing agent, as supported elsewhere [22]. However, in the absence of our catalyst the thermodynamically favorable reduction of 4-nitrophenol was not watched and the absorbance peak corresponding to 4-nitrophenolate ions at 400 nm rest unchanged for a long time (Fig.6-B). Then, when a small amount of Ag 3 PO 4 nanostructured (5 mg) was introduced into reaction solution, the absorbance peak at 400 nm decreases signi cantly within 32 min and concomitant appearance of a new peak at 300 nm. The new absorption at 300 nm is characteristic peak of 4-AP, revealing the reduction of 4-NP to form 4-AP. In addition, as seen in the UV-Vis spectra ( Fig.6.C), the presence of an isobestic point at 317 nm indicating that the catalytic reduction of 4-nitrophenol gives 4-aminophenol only without by product [23][24].
To thus, an optimum concentration of 0.1M [NaBH 4 ] was chosen for the future experiments. In addition, the effect of amount of the Ag 3 PO 4 nanostructured on catalytic e ciency was also studied using 0.1M of NaBH 4 at room temperature. We should mentioned that the reaction was started after adding of Ag 3 PO 4 as a nanostructured catalyst and the colour of the solution changed gradually from bright yellow to colourless indicated the successive reduction of 4-NP. As showed in Fig. 7b, the conversion reaction seems to be sensitive to the catalyst amount, but from 5 mg of the catalyst, the reaction became uncontrollable and ends very quickly. Thus, the optimum amount of the catalyst was selected to be 5 mg.
Based on the results described above the proposed mechanism for the reduction of 4-NP is given in schematic 3. As published elsewhere [27][28][29], the hydrogen atom of BH 4 is positively charged and could create ne electrostatic attractions with negatively charged oxygen from nitro groups at catalyst surface, facilitating the removal of oxygen and reduction of nitro groups. In addition, the residual nitrogen of -NO 2 is also negatively charged due to its greater electronegativity than carbon from the benzene ring, and the H atoms of the positively charged H 2 O molecules could easily combine with the residual nitrogen of the nitrophenol to form the nal aminophenol product. Adding to the proton transfer and deoxygenation, electron transport must occur simultaneously from the BH 4 clusters to 4-NP via the Ag 3 PO 4 catalyst substrate to compensate for the charge balance and accomplish the process of reduction.
The reusability of the catalyst is another important factor from economic and environmental point of view, which it is highly desirable to examine in this study. At the end of the reaction, the catalyst was easily separated by ltration from solution, washed with deionized water and ethanol, dried at 80°C and then was reused for the next cycle of catalysis. As shown in Fig. 8, the catalyst was recycled several times with a little loss in catalytic performance after the third cycle. This can be explained by the reduction of silver (Ag + →Ag 0 ) by the excited electrons during catalytic processes, con rmed by the gradual colour change of the Ag 3 PO 4 catalyst (From yellow to dark brown), resulting in decrease in the catalytic e ciency.

Antibacterial activity studies
The production of a large quantity of Ag 3 PO 4 through a simple and economical method from natural phosphate can be employed as antibacterial agent suitable for the biological treatment of wastewater. In this optic, the obtained results of antibacterial activity of Ag 3 PO 4 against E. Coli (Gram-negative) and S.
Aureus (Gram-positive) are shown in Fig. 9. The zone of inhibition clearly indicated the signi cant antibacterial effect of the nanostructured Ag 3 PO 4 as quantitatively shown in Table 2. With 1 mg/mL as the serial concentration of Ag 3 PO 4 in the biological solution, the maximum diameters of the zones of inhibition are approximately 12.01 mm and 13.50 mm against S. aureus and E. coli, respectively. As result, the nanostructured powder of Ag 3 PO 4 prepared from phosphate rock is in fact an effective antibacterial agent on Gram positive and Gram-negative bacteria such as largely described in the literature.

Conclusion
This study develops a novel approach for the synthesis of the single phase of the nanostructured Ag 3 PO 4 from natural phosphate as phosphate source via dissolution-precipitation process. The as-prepared sample was successfully characterized by various physicochemical techniques in order to study its thermal, structural, textural and morphological properties. Then, the nanostructured catalyst exhibited higher catalytic activity towards the reduction of 4-nitrophenol to 4-aminophenol using NaBH 4 as reducing agent in aqueous solution. In addition, the prepared Ag 3 PO 4 catalyst possesses signi cant antibacterial activities against E. Coli and S. Aureus bacteria. The operational simplicity, short reaction times, recyclability and antibacterial activity are the outstanding features of the present study.

Declarations
Funding: The authors would like to thank the Moroccan Foundation for Advanced Science, Innovation and Research (MAScIR) for its nancial support (allowance) particularly for giving us the opportunity to have access to its fully sophisticated technological platform to perform this work and to characterize our materials.   FT-IR spectra of nanostructured Ag3PO4 powder.   Reuse performance of nanostructured Ag3PO4 catalyst in reduction of 4-nitrophenol to 4-aminophenol.

Figure 9
Antibacterial test results for E. Coli and S. Aureus after 24 hours of incubation using nanostructured Ag3PO4 (1mg/mL). Scheme 1. Schematic diagram of the preparation of nanostructured Ag3PO4 by dissolution-precipitation process from natural phosphate.