Green Synthesis of Nano-Sized Sulfur and Its Effect on Plant Growth

The major objective of this research paper is to synthesis sulfur nanoparticles (SNPs) by green route, which can improve plant’s growth and reduce the environmental pathogens. Sulfur nanoparticles (SNPs) were synthesized by a simple green procedure using Melia azedarach leaves aqueous extract and citric acid. The leaves of Melia azedarach aqueous extract act as a capping and stabilizing agent in the formation of sulfur nanoparticles. The synthesized sulfur nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). The average particles diameter size was found to be 20 ± 4 nm. Analysis showed that the nanoparticles are crystalline in nature, with spherical shape. A preliminary field study was conducted to evaluate growth and development of Cucurbita pepo in response to sulfur nanoparticles.


Introduction
Nanotechnology has gained intense attention in the recent years due to its wide application in diverse areas like medicine, catalysis, energy and materials.Particularly nanoparticles with small size to large surface area have potential medical as well as industrial applications.Researchers make significant efforts towards the synthesis of nanoparticles by various means, including physical, chemical and biological methods.Green methods for synthesizing nanoparticles with plant extracts are advantageous over physical, chemical and microbial methods as it is simple, convenient, environment friendly, and requires less reaction time.Sulfur has a wide range of applications in different agriculture area.Sulfur can be used as fungicide against many plant diseases such as the apple scab disease in the cold conditions, also Sulfur used in the culture of grapes, vegetables, strawberry and many cultivated plants.However, sulfur can be considered as a high efficiency pesticide that used in agriculture where it has good effect against a wide range of powdery mildew diseases as well as black spot.Sulfur nanoparticles have many advantages over micro-sulfur for their peculiar quantum size properties and high surface areas.Recently, great effort has been focused on finding approaches for synthesis sulfur nanoparticles (SNPs) with well-defined shapes and nano-sizes.Among these, sulfur nanoparticles synthesized from H 2 S gas by using biodegradable iron chelates catalyst in reverse microemulsion technique (Deshpande et al., 2008;Guo et al., 2006), an electrochemical method using thiosulfate ion (Shamsipur et al., 2011), water-in-oil microemulsion system using cyclohexane as oil phase, butanol as co-surfactant, nonionic surfactant Triton X-100 and sodium polysulfide (Soleimani et al., 2013), an aqueous solution of potassium polysulfide and various organic and inorganic acids (Massalimov et al., 2012), sublimed sulfur in ethanol using ultrasonic technique (Xie et al., 2009), dissolving sublimed sulfur in a green solvent PEG-400 (Xie et al., 2012), aqueous surfactant (Chaudhuri andParia, 2012), supersaturated solvent method (Wu et al., 2008), heating sulfur powder with polyethylene glycol PEG-600 (Meenatchi et al., 2015), and eggshell membrane as natural biomaterial (cheng et al., 2011).These methods have many disadvantages due to the difficulty of scale up of the process, separation and purification of nanoparticles from the microemulsion (oil, surfactant, co-surfactant and aqueous phase), and consuming huge amount of surfactant.In previous work, sulfur nanoparticles synthesized by green method from sodium thiosulfate in presence of Albizia julibrissin fruits extract (Awwad et al., 2015).As continuation of our previous work, we report here for the first time a novel, rapid, cost-effective and environmentally biosynthesis of sulfur nanoparticles using Melia azedarach leaves extract at room temperature.The major objectives of this research is to synthesis sulfur nanoparticles by an environmentally green method, which can improve plant's growth and reduce the environmental risks as those caused by pathogenic organisms.

Preparation of Melia Azedarach Leaves Extract
Fresh leaves of Melia azedarach were collected from the campus of Royal Scientific Society, Jordan.Fresh leaves were washed under running tap water to remove any debris and dust attached to the leaves and subsequently with distilled water 3-4 times.After washing the leaves were separated by cutting them into small pieces and air dried for four weeks at room temperature.The dried leaves were finely powdered through grinding using Restch RM 100 grinder.The extract was prepared by taking 20 g of powdered leaves and boiled in 500 ml glass beaker along with 400 ml of sterile distilled water for 10 minutes.The aqueous extract of leaves was separated by filtration with Whatman No. 1 filter paper and then centrifuged at 1200 rpm for 5 minutes to remove heavy biomaterials.The filtrate was collected and stored at room temperature.This gave the final leaves extract which was used for further experimental procedure.

Synthesis of Sulfur Nanoparticles
In a typical reaction synthesis, sulfur nanoparticles (SNPs) synthesized as follows: An appropriate amount of sodium thiosulfate pentahydrate (Na 2 S 2 O 3 .5H 2 O) was dissolved in 100 ml of Melia azedarach leaves extract under mild stirring for 5 minutes at room temperature and then diluted with deionized distilled water to 500 ml.Afterwards citric acid (0.5 g/ml) was added drop by drop under stirring for allowing the sulfur precipitations uniformly.The suspended sulfur particles obtained were separated by centrifugation at 1000 rpm/min for 5 minutes and then repeatedly washed with sterile distilled water to remove any biological materials.Sulfur nanoparticles after purification were dried in a vacuum at 80 o C for 6 h.The product was light yellow powder used for SEM, XRD, and FTIR analysis.

Characterization Techniques
Crystalline sulfur nanoparticles were examined by X-ray diffractometer (XRD-6000 Shimadzu, Japan) equipped with Cu Κα radiation source using Ni as filter and at a setting of 30 kV/30 mA.All XRD data were collected under the same experimental conditions, in the angular range 3 o ≤ 2θ ≤ 60 o .Fourier transmission infrared (FT-IR) spectra for Melia azedarach leaves extract and sulfur nanoparticles were obtained in the range 4000-400 cm -1 by IR-Prestige-21, FT-IR spectrophotometer (Shimaduz, Japan) using KBr pellet method.Scanning electron microscopy (SEM) analysis of sulfur nanoparticles was done using Quanta FEI 450 SEM machine.

Plant Growth and Sulfur Nanoparticles
Experiments were conducted in 10 m × 10 m greenhouse compartments, which were part of the Department of Plant Protection, Faculty of Agriculture, the University of Jordan.In all experiments, the indeterminately growing round Cucurbita pepo was used.After 10 days sowing in trays filled with a commercial potting soil, plants were picked out and transferred to a field at the Royal Scientific Society, where the grown in soil of the field.The response of C. pepo to sulfur nanoparticles was studied through field experiments conducted during April-May 2015.Sulfur nanoparticles (SNPs) were applied to the field soil 100 ppm-600 ppm doses.Plant height was measured from bottom to top of C. pepo.Stem diameter was measured from base, middle and top portion and average was taken.Number of branches and leaves were counted.

X-Ray Diffraction Analysis (XRD)
The XRD analysis of the synthesized sulfur nanoparticles is illustrated in Figure 1 are attributed to the crystal planes of sulfur.The sulfur nanoparticles are well-crystalline and the relative intensity of the diffraction peaks match well with the standard monoclinic phase sulfur diffraction pattern (JCPDS 08-0247).The average particle sizes of the synthesized sulfur nanoparticles were calculated using Debye-Scherrer formula (Klug & Alexander, 1954): Where D i lines, λ is K is the S related to sulfur nano

Acknowle
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