Recently, in the presence of extracellular or intracellular extracts of microorganisms such as bacteria, fungi, and yeast strains, the biosynthesis of nanostructures has attracted attention due to its ease of preparation, photoelectric and physicochemical properties; in addition, it has excellent antimicrobial activity^1-4. Although the reduction rate of metal ions in nanoparticles is faster, the existence of extracellular extracts (i.e. plants and microorganisms) is much faster under environmental conditions of temperature and pressure^5-7. Several studies have been devoted to improving the synthetic efficiency such as control of particle size, shape, and mono-dispersity^8-10. Zinc oxide (ZnO) is an inorganic metal oxide that has a wide range of applications, such as medicines and cosmetics, and is mainly used to fight infections in the form of ointments and creams. Similarly, ZnONPs combined with other metal oxides (such as SiO₂ NPs) is used as UV blockers in textiles and induce superhydrophobicity and bacterial growth inhibitory properties^11-13. In addition, ZnO NPs are widely used as an additive in ceramics, glass, cement, rubber, lubricants, paints, ointments, adhesives, pigments, foods, batteries, first-aid tapes, etc.^14,15. Moreover, ZnO NPs have higher catalytic efficiency, and chemical stability, the antimicrobial mechanism of ZnO NPs is still open and not well known. Some authors suggest that ZnO NPs didn’t exhibit any antimicrobial activity by themselves, but enhances the antimicrobial activity of certain antibiotics¹⁶. The antimicrobial effects of ZnO NPs against many foodborne pathogens have been reported, such as E. coli O157:H7¹⁷, C. jejuni¹⁸, P. aeruginosa¹⁹, Salmonella spp.²⁰ and Staphylococcus aureus²¹. In addition, ZnO-NPs have antibacterial efficacy against both the Gram-negative and Gram-positive bacteria and many viruses such as Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtillis, B. atrophaenus, Salmonella typhimurium, Klebisella pnumoniae, Alfalfa mosaic virus, etc.^22-26. Furthermore, ZnO-NPs have anti-fungal activity against several types of molds such as Aspergillus flavus and A. fumigatus and many other microscopic molds, such as Botrytis cinerea, Penicillium expansum, Phanerochaete salmonicolor, Candida albicans, and Fusarium oxysporum^27,28. Up-to-date, some studies have been devoted to reporting that fungal-mediated synthesis is more advantageous because fungi are super accumulators, showing economic viability and easy expansion of synthesis^29,30. Furthermore, a large number of extracellular proteins and enzymes have the dual function of producing and coating mono-disperse nanoparticles³¹. In addition, fungi are very tolerant of higher concentrations of metals and, due to the presence of a large number of extracellular proteins and redox enzymes; they have a large number of functional groups that can reduce metal ions to zero-valence metal nanoparticles³². The cytotoxic mechanism of ZnO-NPs is not yet fully understood, but hydroxyl radicals (OH^•), superoxide anions (O₂^–), and per-hydroxyl radicals (HO₂^•) generated from the surface of ZnO are believed to be the main components. When Nanoparticles interact with cells, cellular protection mechanisms are activated to minimize damage; however, if the production of highly active free radicals more exceeds the cell’s antioxidant defence capabilities, biomolecules will undergo oxidative damage, leading to cell death^33,34.

In this study, green ZnO nanoparticles have been synthesized by using extracellular proteins and enzymes present in Aspergillus niger extract to reduce Zn²⁺ present in zinc acetate as a metal precursor. In addition, antimicrobial agents will be studied, including antibacterial and fungal activity against bacterial and fungal species.

Experimental Section
Materials
Zinc acetate salt (Zn (CH₃COO)₂) was pursued from Sigma-Aldrich, Whatman filter paper. Glassware was washed with sterile distilled water more than one time then dried in an electric oven before using to remove any contaminations. Muller-Hinton agar³⁵, Nutrient Agar medium³⁶. Nutrient Broth medium³⁷, Potato Dextrose Agar (PDA)³⁸ and Potato Dextrose Broth (PDB)³⁹, from Sigma Aldrich- Germany.

Methods
Isolation and purification of fungal isolates
Soil samples were taken from the garden of the Botany and Microbiology Dep., Faculty of Science, Al-Azhar University, Cairo, Egypt, at approximately 10 cm depth. One gram of soil sample collected was suspended in 100 ml of distilled and sterilized water. One milliliter (1 ml) from 10^-1 to10^-6 dilutions of soil suspension of the sample was placed on the plate⁴⁰, with media Czapex-Dox Agar (CDA) media g/L: Sucrose: 30g. NaNO₃: 2g. , KH₂HPO₄: 1g. KCl: 0.5g. MgSO₄.7H₂O: 0.5g, FeSO₄.7H₂O: 0.01g, Agar: 20g, and distilled water: 1000 ml, pH7, supplemented with chloramphenicol 0.5% to suppress bacterial growth⁴¹. A triplicate of plates was used for each particular dilution. Then, the plates were incubated for seven days at 27±2°C until colonies appeared. The isolation of fungi was carried out by using a CDA medium according to the method described by⁴².

Identification of most potent fungal isolate
Morphological, cultural and microscopic examination for fungal isolate
A Purified fungal isolate was identified based on routine cultural and morphological characteristics. Fungal genera and species were identified according to standard manuals^43,44. Macroscopic examinations were carried out to study the culture characteristics, including colony surface color, reverse color, colony growth rate, and pigmentation.

Molecular identification for fungal isolate
Use the Easy Pure Genomic DNA Extraction Kit to isolate DNA samples from the tested fungal strains, and use Nano-Drop to measure the purity and appropriate concentration of the DNA samples. Four samples of Aspergillus niger DNA for PCR reactions. Two fungal universal primers, FW 5-ATGGGCAAGGCACCAAATAA-3 and RW 5-TGGAAATGGATC CAAGAATG-3 were used to carry out the PCR reaction of 4 fungal 18s rDNA gene amplification. The PCR reaction conditions were adjusted to 40 ng (6 µl) DNA template (fungi) and 8.5 µl master mix, which includes a mixture of dNTP, MgCl₂, Taq polymerase, and PCR buffer. Add primers separately after preparation from the lyophilized stock solution (each primer is 1 μmol/l). The PCR conditions were adjusted for the denaturation step of 92.3°C, the hybridization step of 55.6°C and the extension step of 71.9°C. The number of PCR cycles was 36 cycles, and the El-Dokki PCR thermal cycler from the National Research Center (NRC) of Giza, Egypt was used. Use a 1% agarose gel to separate the amplified DNA product and, DNA markers [Gene Ruler 100 bp DNA Ladder (SM0241)] by electrophoresis. The gel was stained with ethidium bromide and the band profile was recorded using the UV gel documentation system. Purify, and sequence the amplified DNA products. The 4 sequences were analyzed and tested against the most closed sequence in Gen Bank NCBI through BLST, and the phylogenetic tree was designed using MEGA 7 software.

Preparation of fungal biomass extract
A Purified fungal isolate was tested for ZnO NP biosynthesis by growing in Czapex Dox broth (CBD)⁴⁵. Two discs (0.7 mm) of purified fungal isolates were freshly incubated in 50 ml of CBD in a 250 ml Erlenmeyer flask as a fermentation medium. Incubate at rpm for 6 days. The fungal biomass is collected by passing through two layers of medical gauze and washed with distilled and sterile water to remove any adhering medium components. Then, ten (10) g of fungal biomass were suspended in 100 ml of distilled water in a 250 ml Erlenmeyer flask and stirred at 150 rpm at 28 ± 2°C for 72 hours. Then, the cells were discarded by Whatman No. 1 filter paper to obtain a biomass filtrate for nanoparticle synthesis.

Extracellular green synthesis of Zinc oxide nanoparticles using fungal isolates
Typically, in a 250 ml reaction vessel, an equi-volume ratio (1:1) aqueous solution of zinc acetate (1 mM/100 ml, Zn (CH₃COO)₂) was added to 100 ml of fungal extract filtrate under orbital shaking at 150 rpm. The reaction was incubated at pH ~ 6.5, and a temperature of 32°C for 72 hr. in dark conditions. A white precipitate is formed indicating to the formation of ZnONPs. The as-prepared ZnONPs were washed several times with sterile distilled water then centrifuged at 10,000 rpm for 10 min⁴⁶.

Characterization
Use the JASCO 730 Double Beam Spectrophotometer to obtain the optical UV-Vis absorption characteristics. The absorption spectrum was recorded in the range of 200 to 900 nm and the increase in wavelength was approximately 0.2 nm. The JEOL Transmission Electron Microscope (TEM), model 1200EX, is used to study micrographs of samples obtained at an operating voltage of 120 kV. In addition, the Ultima IV powder diffractometer (Rigaku) has been used for X-ray diffraction (XRD) measurements in the 2 rango range of 20-70 degrees using a Cu objective with Kα1 = 1.54060 Å. X-ray scanning is performed in a 2θ / θ continuous mode at a speed of 2 degrees per minute with a step size of 0.02. The tube voltage and tube current are maintained at 40 kV and 40 mA. It Uses the Malvern Zetasizer Nano (ZS) instrument and He/Ne laser (633 nm) to collect backscattering optics at an angle of 173° to measure the size distribution and zeta potential of the sterilized ZnONP. In addition, according to Vivek et al.⁴⁷, the JASCO 6700 Fourier Transform Infrared Spectrometer (FT-IR) was used to obtain the FT-IR of green synthetic ZnONP in the range of 400 to 4000 cm^-1.

Antimicrobial property
Tested microorganisms
Bacteria
Six isolates, Klebsiella pneumoniae, Escherichia coli, Acinitobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus haemolyticus were isolated from clinical samples and identified based on culture, morphology, and biochemical analysis according to Bergey’s manual⁴⁸. In addition, a Vitek2 system was carried out to confirm the identity.

Fungi/Yeast
Four isolates were Aspergilus niger, Penicillium marneffei, Candida glabrata, and Candida parapsilosis isolated from clinical samples and identified in the Mycology Lab. of the Botany and Microbiology Dep. Faculty of Science, Al-Azhar University, Cairo, Egypt.

Antimicrobial Experiments
The zinc oxide nanoparticles (ZnO-NPs) synthesized from Aspergillus niger biomass were tested for antimicrobial activity via two methods; the agar-well diffusion method and the broth micro-dilution assay.

Agar well diffusion method
Apure cultures of the tested bacteria were sub-cultured in nutrient broth and each strain was uniformly spread on sterilized petri plates with Muller-Hinton agar. A circular well of 6 mm in diameter was made in plates using a sterile cork-borer. Each well was loaded with (50 µl) ZnO-NPs to check the antibacterial activity and the plates were incubated at 37°C overnight and the zones of inhibition were measured. Additionally, the antifungal activity of zinc oxide nanoparticles was examined against tested fungal species. The strains were maintained on CDA at 28°C and 5-d old cultures were used for antifungal analysis. Pour three to four milliliters of sterile normal saline onto the fungal growth; gently scrape to collect the conidia. Spread 100 μl of this liquid spore suspension evenly on a fresh potato dextrose agar (PDA) plate. Use a sterile cork-borer to make a 6 mm in diameter circular hole in the plate. Each well was filled with (50 µl) zinc oxide nanoparticles to check antifungal activity, then incubated the plate at 25°C for 2-3 days and measured the zone of inhibition⁴⁹.

Broth microdilution assay
The suspension was turbid to 0.5 McFarland standards (10⁸ cfu / ml) produced by fresh subcultures of bacterial and fungal/yeast Mueller Hinton Broth (MHB) and Potato Dextrose Broth (PDB), respectively. The corresponding suspension was diluted to 10⁶ cfu / ml. Prepared microbial inoculation (100 μl) was added to each well of a sterile flat bottom 96-well microtiter plate containing the tested concentrations of ZnO-NP (100 μl/well). As a result, a final inoculation concentration of 5x105cfu/ml was obtained from each well. The tested ZnO-NP used in the growth control of the tested microorganisms was contained in wells containing microbial suspensions and other well plates containing double background control. The optical density was measured at 620 nm after 24 hours at 37°C for bacteria, 48 hours at 28°C for mold / yeast, using an ELISA microplate reader (Sunrise ™ -TECAN, Switzerland) at the Faculty of Science Al-Azhar University in Cairo, Egypt. Finally, the cell concentration was converted to an average growth inhibition percentage (%). The rate of decrease in microbial growth (GR %) was estimated as follows, based on the treatment of the control group (excluding the ZnONPs). GR% = CT / C x100 where C is the treated cell concentration of the control group and T is the ZnONPs process. Three replicas were considered. The Results were reported as the mean ± SE of the experiments⁵⁰.

Preparation of Resazurin Solution
The concentration of resazurin stain solution is 0.02% (w/v)⁵¹. Dissolve 0.002 g of resazurin stain salt fine powder in 10 ml of sterile distilled water and vortex. The complete mixture is filtered through a Millipore membrane filter (0.2 µm). This solution can be stored at 4°C for 2 weeks.

Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration of biosynthesized ZnO NPs was evaluated against bacterial and fungal strains via a method described in the guide⁵⁰. The MIC test is evaluated on a 96-well microtiter plate with a round bottom by a standard broth microdilution assay.

For bacterial strains; the concentration of the bacterial inoculum was adjusted to 106 cfu /ml. Add 100 μl of biosynthesized ZnO-NPs stock solution (400μg/mL) to the first well, dilute 2 times with the bacterial inoculum, and use 100 μL media MHB from column 4 to column 12. The fourth column of the plate contains the highest concentration of ZnO NP, while the 12th column has the lowest concentration. Column 1 serves as a positive control (medium and bacterial inoculum), and column 2 serves as a negative control (medium only). Add 30 μl resazurin solutions to each well of the microtiter plate and incubate for 24 h at 37°C. A color change is observed. Blue/purple means no bacterial growth, while pink/colorless means bacterial growth. The MIC value is obtained at the lowest concentration of the antibacterial agent that inhibits the growth of bacteria, the color remains blue.

For fungal strains, a suspension of the original inoculum in sterile saline containing 1% Tween 80 (supplied by Trek Diagnostic Systems) was prepared from 7-day old cultures grown on a PDA slope. For an inoculation density of 95%, the actual stock inoculation suspension calculated by quantifying the inoculation varies between 0.9 × 10⁶ and 4.5 × 10⁶ cfu / ml. One hundred (100 μl) biosynthesized ZnO NPs stock solution (400μg/mL) was added to the first well of another new plate (96-well microtiter plate with a round bottom). Followed by a 100 μl of fungal conidia inoculum suspension in liquid potato dextrose agar (PDA) was added. The microdilution tray is incubated at 30°C and checked after 4 days of incubation. The endpoint of MIC is the lowest concentration of ZnNP showing no growth or complete inhibition of growth (100% inhibition)⁵². On the other hand, as described above, unicellular fungi are evaluated as bacteria.

Cytotoxicity assay to evaluate nanoparticles toxicity by using tissue culture
According to Riss and Moravec⁵³ (MTT protocol), the culture plate with (96-well tissue) was cultivated with 1×10⁵ cells/ml (100 μl/well) and incubated for 24 hours at 37°C to form an integrated monolayer. After forming a collected cell sheet, pour out the growth medium from the 96-well microtiter plate, and the cell monolayer was washed twice with a washing medium (duple dilution) in a medium containing 2% serum (maintenance medium). 0.1 ml of each dilution test was prepared in different wells, 3 wells were left as controls, and only contained medium. Incubate the plate at 37°C. Check the cells for any changes that indicate physical toxicity, such as partial or complete damage of monolayer, shrinkage, rounding, or granulation of the cells. 20ul of MTT solution was added to each well, then shacked for 5 minutes at 150 rpm to optimum mix the MTT in the medium completely, then incubated for 1-5 hours at 37°C and 5% CO₂ to let the MTT metabolize. Then remove the medium, dry the towel if necessary to remove debris, resuspend the formazan (metabolic product MTT) in 200 ul DMSO, and shake it for 5 minutes at 150 rpm to remove the formazan mixed thoroughly into the medium. Finally, record the optical density at a wavelength of 560 nm and subtract the background at 620 nanometers. The optical density must be directly related to the number of cells.

Statistical analysis
All the experiments were performed in triplicate and data were analysed. Analyses were performed as prescribed by Kareem et al.⁵⁴.