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1-The work was initiated by screening of twenty different fungal species isolated in this work from soil samples collected from some localities in Giza and Dakahlia governorates of Egypt to examine their potentiality for the biosynthesis of extracellular CuO NPs using culture supernatant (CS) and/or washed biomass of the isolated fungi.
2- Of the screened fungi, Aspergillus fumigatus Fresenius AUMC 13024, was confirmed for the first time as far as the author is aware the suitable source for biosynthesis of CuO NPs using its preformed biomass. Such organism was isolated from soil sample collected near Giza city.
3- When the biomass was contacted with 1 mM solution of copper nitrate and incubated in shaking incubator, color of the reaction mixture was changed from light blue to light green in a preliminary indication of CuO NPs biosynthesis which was then confirmed by UV-Vis spectroscopy. A. fumigatus biomass had got the power to synthesize relatively good yield of the CuO NPs with an intense peak at 335 nm.
4- Biosynthesis of the investigated NPs by A. fumigatus biomass was carried out on five different fermentation media. The results demonstrate fluctuation of the fungus behavior on these media. The highest potentiality towards the biosynthesis of CuO NPs was recorded in case of Czapek’s medium.
5- A series of experiments were conducted to investigate the role of some reactions conditions affecting the biosynthesis of CuO NPs using the preformed biomass of A. fumigatus.
a- pH value: The maximal production of the NPs was recorded at pH 6.
b- Temperature: The highest synthesis was revealed at 30oC.
c- Precursor salt: Nitrate salt (Cu(NO3)2.3H2O) was found superior to the sulfate salt (CuSO4.5H2O) in this respect.
d- Copper nitrate concentration: Increasing the concentrations from 0.5 mM to 1 mM greatly enhanced the yield of the NPs. Any further increase depressed the biosynthetic process and at 5 mM no NPs were produced.
e- Incubation period: Production of the NPs was initiated sluggishly, but starting to be visible after 24 h with good absorption at 335 nm. The maximal yield was recorded after 60 h of incubation.
6- To indicate the possible involvement of various functional groups or biomolecules in the formation and stabilization of CuO NPs, the FTIR spectroscopy of CuO NPs biosynthesized by A. fumigatus biomass was carried out. The FTIR spectrum showed many functional groups that plays an important role not only in biosynthesis of CuO NPs but also in capping them.
7- Characterization of the biosynthesized CuO NPs was achieved using HR-TEM, DLS, XRD and EDX.
a- HR-TEM: HR-TEM micrograph showed morphology and size details of the biosynthesized CuO NPs. These NPs were spherical in shape, good distributed with no signs of agglomeration. They exhibit variation in size but the predominant were those of the sizes 4-16 nm. The NPs had clear edges suggesting their highly crystalline nature. This suggestion was sustained by the SAED pattern where the diffraction rings appeared as lighted spots on the dark field
b- DLS: The average size of CuO NPs was estimated by DLS to be 96.2 nm with a polydispersity index (PDI) of 0.639. The variation in the size estimate between the DLS and TEM is due to the fact that the DLS method measures the whole diameter size of the NPs and their capping molecules at the time that the TEM measures only NPs diameter.
c- Zeta potential: Zeta potential value of the biosynthesized NPs was calculated to be -28.2 mV. The negative charge was suggested to increase stability of the NPs which maintained their characteristics for at least six months of storage at 4oC.
d- XRD: Crystalline nature of the produced CuO NPs was illustrated by their XRD analysis. Eleven Bragg diffraction peaks at 2θ were recorded agree with the values reported for CuO NPs (JCPD file number 45-0937).
e- EDX: The EDX pattern of the produced CuO NPs revealed the presence of the signal characteristic of copper (Cu) and oxygen (O) elements in weight percent of O: 12.09 weight % and Cu: 87.91 weight %, respectively.
8-The second part of this work includes the study of some possible biomedical applications of CuO NPs biosynthesized using the preformed biomass of A. fumigatus:
Antimicrobial activity of the biosynthesized CuO NPs against four different bacterial strains including human pathogens, two Gram positive i.e. B. subtilis ATCC 6633 and S. aureus ATCC 8538 and two Gram negative i.e. E. coli ATCC 8739 and K. pnemonia ATCC 27736 was investigated qualitatively by the disk diffusion method and quantitatively in term of MIC. The results demonstrate a fluctuation effect of the NPs on different test bacteria and this can be attributed to the difference of cell wall composition. MIC was varied between 50µg/ml for the tested Gram +ve and 60µg/ml for the Gram –ve bacteria. Presence of CuO NPs can induce the generation of free radicals and formation of oxidative stress leading for damage of bacterial cell wall and cell membrane. The results showed that intracellular components like sugars, proteins and nucleic acids were leaked from the bacterial cells after their contact with CuO NPs. Leakage of these components was a clear indication for the bacterial cell wall damage owing to the presence of NPs that finally lead to cell death.
b- Antioxidant and free radical scavenging activity (RSA):
The RSA of the biosynthesized CuO NPs using A. fumigatus preformed biomass was examined in vitro using different free radical scavenging assays. The results reveal that the RSA of these NPs was concentration-dependent manner. They had good scavenging activity against DPPH, hydroxyl, peroxide and superoxide radicals. Different RSA of these NPs can be compared favorably with those of the standard antioxidants. The antioxidant activity of NPs is may be due to the preferential adsorption of the antioxidant material onto their surface.
A confirmation for this suggestion was obtained from the FTIR analysis where phenolic hydroxyl compounds and other chemical compounds that serve as antioxidants have been found in attachment with the surface of CuO NPs.
c- Photocatalytic biodegradation activity:
The CuO NPs biosynthesized in this work was investigated as a tool for the photocatalysis of methylene blue under direct sunlight and found that 97 % of that dye was degraded after 200 min. Three main components are involved in the degradation process: light, CuO NPs as a catalyst and oxygen. This technique seems to be the most practical process for complete mineralization of these pollutants due to its low cost with no extreme conditions and nil toxicity.