Synthesis of α-Al2O3 Nanoparticles From Pepsi Cans Wastes and Its Fungicidal Effect on Some Mycotoxins Producing Fungal Isolates

Green synthesis of nanomaterials is the most recent trend in nanotechology because it is synthesized the highly valuable compounds (nanomaterials) from wastes to reduce the highly negative impact of these wastes in environment. The aim of this study was to isolate and purify the most common mycotoxin, aatoxin producing fungi from Maize and soybean grains which considered as the great economical importance as both animal and human feed. In addition to green synthesized of a-Al 2 O 3 from cans and to detect its antifungal effect on the isolate’s growth at different concentrations. Thereafter, determining the fungicidal concentration of the tested nanoparticles on the isolated fungal strains. The structural, morphological, optical and antifungal activity of the prepared a-Al 2 O 3. are characterized using X-ray diffraction (XRD), High resolution transmission electron microscope (HRTEM), Field Emission Scanning Electron Microscope (FESEM), Attenuated total Reection-Fourier Transform Infrared (ATR-FTIR) and Ultraviolet-Visible (UV-Vis) spectrophotometer. Results shows that, the most common fungal strains presented were belonging to Aspergillus avus, Fusarium oxysporum and Alternaria sp.. Formation of Al 2 O 3 is conrmed using XRD, FESEM and ATR –FTIR. The average particle size of a-Al 2 O 3 is 4-10 nm. Optical band gap of a-Al 2 O 3 are calculated using Tauc relation. Through investigating the fungicide concentration, Data showed that the maximum antifungal activity of aluminum nanoparticles a-Al 2 O 3 was detected for A. avus, Fusarium sp.and Alternaria sp. in concentration 1, 6 and 50 mg/100ml respectively.


Introduction
In recent years, Scientists have recently turned to green synthesis of nanoparticles from wastes and used these in many applications. The valuable use of nanoparticles is their superior antimicrobial e cacy as compared to their bulk counterparts. There is a growing need to develop new and effective antimicrobial agents as the threat of bacterial and fungal infections grows.
Nanoparticles have also been used in food preservation, burn dressings, medical equipment, water treatment, and a variety of other items in this direction [1,2].
Wastes come from Aluminum cans makes a great problem in the environment. So, recycling of this type of waste save money, energy and time in addition to the money that comes from recycling cans helps people and their communities [3][4][5]. Due to its distinct physical and chemical properties, alumina has a wide range of industrial applications.
Aluminum oxide comes in a variety of types, all of which are produced by calcining the aluminum hydrates bayerite, gibbsite, and boehmite [6]. α-Al 2 O 3 nanopowder is one phases from many type of alumina and it take attention due to it is high chemical resistance, high abrasion resistance and high thermal stability [7]. Due to a favorable combination of properties such as high mechanical strength and hardness, strong wear resistance, high refractoriness, and high corrosion resistance in a wide variety of chemical environments, α-Al 2 O 3 is one of the most commonly used ceramic materials [8].
In developing countries, Maize and soybean grains are considered major crops for human and animal feed due to its relatively high protein and suitable amino acid concentration that simultaneously affect the health, breeding and productivity. Inappropriate preservation of Maize and soybean usually causes the yield to be infected till spoilage with saprotrophic, mycotoxin producing fungi unless treated with fungicide during the storge period. Animal feeding on spoiled grains lead to accumulative, detrimental effect generally on the animal health and preciously on productivity [9]. The most common fungal infection included but not limited to: Aspergillus sp., Alternaria sp. and Fusarium sp. [10]. Different types of mycotoxins are produced by different fungi, A atoxin production relatively distinct to Aspergillus infection, fumonisin is produced commonly by Fusarium species. Other fumonisin related mycotoxins such as tenuazonic acid, alternariol characteristically distinct to Alternaria infection [11].
The application of synthetic fungicide for long term is not ideal due to its high cost, residues and eventually its impact on the environment and general health. Moreover, the increased resistance of the fungal pathogen towards the fungicide is another setback for the wide and continuous application of chemically synthesized fungicide [12].
Recently, the wide application of nanotechnology for the control of vast majority of plant and human infection against different pathogenic microbes, have attracted increasing attentions as substitute for their chemically synthesized counterparts. Nowadays, Nanotechnology plays a major role in the control of plant infection either through limiting agent transmission or disease detection [13,14]. Additionally, nanotechnology is becoming a potential solution for food borne pathogen control and elimination. In precious, Aluminum nanoparticles have been used for multiple environmental applications due to its wide-known, potent antimicrobial characteristics as food preservative substance.
In our research work, we aimed to prepare α-Al 2 O 3 from Pepsi cans and studied their structural, surface morphology and optical using different tools XRD, ATR-FTIR, HRTEM, FESEM and UV-Vis spectroscopy.
Then the antifungal activity of the prepared α-Al 2 O 3 against the isolated fungal species individually to determine the fungicidal concentration that cause the maximum inhibitory effect on the isolated fungal strains growth.

Preparation of α-Al 2 O 3 NPs
Synthesis of α-alumina from Pepsi cans, the materials used were aluminum cans (collected from wastes), HCl (6 N), NaOH (24%) and deionized water. Cut the Pepsi cans into small pieces. After that slowly adding of pieces into 6 N HCl to avoid any risks during the dissolving of aluminum, After the complete dissolved of cans, separation of the solution from any other impurities by ltration using Whatman 42 lter paper. Then adding the 24% NaOH dropwise to the solution till the aluminum hydroxide was obtained. Washed the prtecipitate three times using centrifuge at 1500 g/15min for elimination NaCl.
Finally, The precipitate was dried at 70°C overnight and calcinated at 350°C/3h.
High -Resolution Transmission Electron Microscope (HRTEM) was performed by JEM-2100F electron microscope with accelerating voltage of 200 kV. Field Emission Scanning Electron Microscope (FESEM) with EDX detector was performed using SEM Model Quanta 250 FEG. ATR-FTIR spectral data are collected using Vertex 80 Bruker (made in Germany) at room temperature in the range 4000 − 400 cm − 1 . UV/vis. spectral data collected using Jasco V-630 spectrophotometer in the range 200-1000 nm at room temperature.

Fungal species isolation and identi cation
Maize and soybean grains that show rotting signs were collected for fungal isolation. The rst step involving surface sterilization of the grains by selecting 1 gm of each grain type and soaking in 0.5% sodium hypochlorite for 3 minutes, followed by wishing with sterilized, distilled H 2 O three times and allowed to air drying under aseptic conditions. subsequently, dried grains were grinded in mortar and suspended in 9ml sterilized distilled H 2 O then vortexed till homogeneity. Serial dilutions were made till the 3rd factor, and 1 ml of the 2nd and 3rd dilution were transferred to rosebengal agar petri dishes and incubated at 30 o C for 5 days. After the incubation period, fungal colony developed were transferred to potato dextrose agar dishes and incubated under the same previous conditions. For fungi identi cation, the fungal isolates developed on PDA petri dishes were identi ed via colony morphology, shape, color and medium pigmentation, as well as microscopic examination of the developed spores and mycelium.

2.4.Screening of aluminum nanoparticles' antifungal activity
Aluminum nanoparticles solution was prepared in concentration (2mg/3ml DDH 2 O) and tested for antifungal activity against Aspergillus avus, Fusarium oxysporumand Alternaria sp. 7mm fungal disk of each isolate was cut and inoculated in 10ml distilled sterilized H 2 O then vortexed vigorously till obtain Fungal spore suspensions of 10 5 -10 6 . 1ml of the fungal spore suspension was inoculated in sterilized petri dishes, covered with warm PDA sterilized medium, and mixed homogenously then left till solidi cation. By mean of sterilize tip end, 7mm agar well was made and inoculated with 50ul (33.3 ug/50ul) of the prepared nanoparticles solution. The PDA plates were left in the fridge for 30 minutes to allow the solution diffusion, then incubated at 30c for 5 day. The antifungal activity was detected after the incubation period by measuring the inhibition zone around the well by ruler in mm.

Determination of the fungicidal concentration:
Actively growing fungal discs of 7mm size in diameter were transferred to 100ml sterilized PDB medium. Different concentrations of α-Al 2 O 3 were prepared according to the isolated fungal strain as follows: For A. avus a concentration gradient of (0, 0.5, 1, 2 and 3 mg/100ml), while for Fusarium sp. (0, 0.5, 2, 4, 5 and 6mg/100ml) and Alternaria sp. (0, 5, 10, 20, 30 and 50 mg/100ml) to be suspended separately in the prepared PDB culture medium altogether with the fungal discs. The cultures were allowed to grow on shaker incubator 180rpm at 30 o C for 5 days under ambient light condition. The fungal mycelia were harvested by ltration through desiccated, pre-weighted Whatman lter paper No.1 and dried overnight at The crystal size of both phases of Alumina is calculated using scherrerr equation [15] Where D is the crystal size, λ is the wavelength of X-ray, β (in radian) is the full width at half maximum (FWHM) of the diffraction peak and θ is the Bragg's diffraction angle (in degree) of the peak maximum. The average crystal size of α-Al 2 O 3 is equal 38.8 nm. The crystallite size is belived to be the size of a coherently diffracting domain which isn't always the same as particle size [16].
The dislocation density, S, is a calculation of the extent of defects and vacancies in a crystal that can be calculated using the formula from the crystallite size (D) [17]. S equal 6.64x10 − 4 nm − 2 forα-Al 2 O 3 . Figure 2 shows HRTEM image of α-Alumina using Pepsi cans. As seen in image the presence of a sponge-like mesoporous structure was detected with average particle size 4-10 nm. Figure 3 represents FESEM for preparing α-Alumina using Pepsi cans. As shown in Fig. 3 this treatment, provide spongy structure with porous and homogenous aggregates with very small size. For EDX analysis as seen con rmed the presence of Al and Oxygen in addition to some element like Na and Cl which present in a small percentageeven after a good washing process, In spite of the washing process was so time-consuming.  Figure 5 shows UV-Vis absorption spectra and optical band gap energy of α-Al 2 O 3 . The spectra shows strong absorption peaks at 245 nm for Aluminum oxide [20,21]. This is due to electron photoexcitation from the valence band to the conduction band.

Optical Properties
The direct optical band gap energy (E g ) of α-Al 2 O 3 is calculated using Tauc relation [22]: Where β is constant and α is the absorption coe cient and it is determined using the following relation [23]: Where A and T are the absorbance and the thickness of the prepared sample, respectively.

Fungal isolates characterization and identi cation
Among 15 fungal colonies obtained on rosebengal agar medium, 3 isolates were signi cantly different on morphological basis (colony shape, texture and medium pigmentation) Fig.6. Morphological and Microscopic examination of the mycelium and spores developed on PDA plates con rmed that the three isolates belonging to Aspergillus avus, Fusarium oxysporum and Alternaria sp according to the taxonomy given by Ainsworth and James, 1971 [24] and Alexopoulos 1996 [25]. Our gathered data from the isolation and identi cation experimental method were compatible to the data obtained by Gulbis Figure 7 and Table 1 presents the effect of aluminum nanoparticles on the selected fungal strains growth inhibition in mm. all the fungal strains show variable sensitivity behavior against Al-nanoparticle. The most affected strain with the maximum inhibitory activity was A. avus followed by F. oxysporum with  Aspergillus avus Figure 8 exhibit that, the maximum inhibitory concentration detected for A. avus was recorded in 1mg/100ml for with inhibition percentage (94.8 %) respectively.  Fig. 9 shows that, the maximum inhibitory concentration was detected for α-Al 2 O 3 NPs in similar ratio at 6mg/100ml with inhibition percentage (98.6%).  [27] detected the antifungal activity of mesoporous aluminum nanoparticles against Fusarium Oxysporum and found that the highest antifungal activity was recorded at concentration 400mg/L with maximum inhibition percentage of 78.57% after growth on PDA plates in comparison to the control treatment.

Screening of the antifungal activity
Alternaria sp. Figure 10 posed that, the highest growth inhibitory concentration was detected at 50mg/100ml value with inhibition percentage of 98.75%. The fungicidal effect of aluminum nanoparticles may be explained by multiple cellular site destruction: either due to its direct linkage to protein and enzymes molecules or DNA contact which causes mutation and adversely affect cellular replication. The antifungal activity may also attribute to linkage to the free hydrogen bond on the cellular membrane which alter the lipopolysaccharide layer con gurations that   the effect of α-Al2O3 NPs on the selected fungal strains. Figure 8 shows the optimum fungicidal concentration for α-Al2O3 NPs against A. avus. Figure 9 shows the optimum fungicidal concentration for α-Al2O3 NPs against Fusarium oxysporum. Figure 10 shows the optimum fungicidal concentration for α-Al2O3 NPs against Alternaria sp.