Synthesised NPs: The formation of chemically synthesised Ag NPs was indicated by the clear solution turning into golden yellow which is in accordance with the previous literature13. The formation of biosynthesised Ag NPs from P. granatum and M. acuminata fruit peels was indicated by the formation of a brown-yellow solution in accordance with the literature5,8. Chemically synthesised ZnO NPs was indicated by the formation of white powder14. Biosynthesised ZnO NPs from P. granatum fruit peels were indicated by the formation of a gel-like product4. The formation of biosynthesised ZnO NPs from M. acuminata fruit peels were indicated by the formation of brown colour solution.
Characterization of NPs
UV-visible spectral analysis of Ag and ZnO NPs: The optical properties of chemically and biosynthesised Ag and ZnO NPs were characterized using UV-Visible spectrophotometer. The absorption maxima for chemically synthesised Ag NPs were observed around 422nm which correlates with previous literature in which absorption maxima was observed around 430nm20. The absorption maxima for biosynthesised Ag NPs from P. granatum were observed around 371nm21. The absorption maxima for biosynthesised Ag NPs from M. acuminata were observed around 438nm which correlates with earlier finding in which it was observed at the spectrum range of 426.50 to 452.00nm15.
Previous literature states that ZnO NPs show absorption maxima in a spectrum range of 360-380nm22. Chemically synthesised ZnO showed maximum absorption at 363nm which correlates with the spectral range of ZnO, thus confirming the formation of ZnO NPs. Biosynthesised ZnO NPs from P. granatum showed maximum absorbance around 364nm4. Biosynthesised ZnO NPs from M. acuminata showed maximum absorbance around 313nm, which correlates with the previous literature which was observed around 351nm16. The chemically synthesised and biosynthesised ZnO NPs exhibited a similar range of absorption maxima. Thus, the absorption range for Ag and ZnO NPs was observed between 250-550 nm thus confirming the formation of respective NPs.
X-Ray diffraction (XRD) analysis of the synthesised NPs: XRD analysis was done using Rigaku X-Ray Diffractometer to determine the crystallite size, and lattice strain of the synthesised Ag and ZnO NPs. The XRD graphs obtained was compared with the respective standard XRD graphs of Ag and ZnO NPs to confirm the presence of NPs (Figure 1(a), (b), (c), (d), (e), (f)). Scherrer’s equation was used to calculate the average crystallite size and lattice strain (Table 1).
The various diffraction peak intensities are a result of the polycrystalline nature of the NPs. Previous literature states that the intense reflection at (111 peak) compared to other peaks indicates the growth direction of Ag nanocrystals (Figure 1(a), (b) &(c)). When compared to other peaks, 101 peak intensity was the strongest for chemically synthesised ZnO NPs (Figure1(d)). The peak intensity was the strongest at (100) as compared to other peaks in both P. granatum and M. acuminata biosynthesized ZnO NPs (Figure 1(e), (f)). Due to various size-dependent factors, irregular peak positions, heights and widths are obtained13. Impurities present in the sample gives peaks other than the Ag characteristic peaks14 and ZnO characteristic peaks.
From the XRD analysis done it can be concluded that the size of chemically and biosynthesized Ag NPs were estimated to range around 12 to 20nm and the size of chemically and biosynthesized ZnO NPs were estimated to range around 0.4-24nm.
Characterization using scanning electron microscope (SEM): SEM analysis was done using Zeiss, Ultra 55 Field Emission Scanning Electron Microscope to determine the surface structure of the Ag NPs and ZnO NPs drop-cast sample. SEM images indicated the length (nm) and shape of the synthesised nanoparticles (Table 2).
The SEM images of the chemically synthesised Ag drop-cast sample were nanospheres (Figure 2 (a)), whereas the SEM images of the chemically synthesised ZnO drop cast sample (Figure 2 (g)) were nanoflakes. SEM images of both biosynthesised Ag and ZnO NPs were nanospheres (Figure 2 (c), (e), (i), (k)). SEM images of P.granatum NPs is similar to previous literature23. SEM analysis of synthesised NPs indicated they are spherical and their sizes were in the range of 30-63 nm.
EDX analysis gives qualitative as well as the quantitative status of elements that may be involved in the formation of nanoparticles (Table 3 & 4). EDX analysis had also carried out to determine the chemical purity, elemental composition, and stoichiometry of the synthesised NPs18.The elemental profile of synthesised Ag NPs using BPE shows a higher count of silver, confirming the NPs formation5.
The elemental profile of Ag NPs from P. granatum and chemically synthesised also showed higher count of Ag, confirming the NPs formation. Similarly, EDX analysis of chemically and biosynthesised ZnO NPs showed higher count of zinc oxide, even though other elements like zinc and sodium were present, which confirms the formation of ZnO NPs.
Antimicrobial activity of synthesised NPs:
The antimicrobial assay was carried out by the well diffusion method against different human pathogenic Gram-positive and Gram-negative bacterial species (E. coli and B. subtilis). Both chemically and biosynthesized silver and zinc oxide nanoparticles were used to assess their antimicrobial activity.
The biologically synthesized nanoparticle had found to be more efficient than the chemically synthesized nanoparticle for antimicrobial activity. The maximum zone of inhibition was observed in M. acuminata Ag NPs (28.4 ± 14.66 mm) against E. coli compared to chemically synthesized silver NPs (16.8 ± 8.44 mm). Also, M. acuminata Ag NPs showed a higher zone of inhibition as compared to P. granatum Ag NPs (20 ± 12.17 mm) against E. coli.
M. acuminata ZnO NPs (10.4 ± 0.89 mm) and P. granatum ZnO NPs (12.8 ± 1.79 mm) exhibited higher zone of inhibition as compared to chemically synthesized ZnO NPs (6.4 ± 3.29 mm) against E. coli wherein P. granatum ZnO NPs showed a higher zone of inhibition as compared to M. acuminata ZnO NPs. Biosynthesized Ag NPs are more efficient as an antimicrobial agent as than biosynthesized ZnO NPs against E. coli. Hence it is concluded that biosynthesized NPs are more efficient as compared to chemically synthesized NPs against E. coli.
The antimicrobial properties of chemically and biosynthesised silver and zinc oxide NPs against E. coli had analysed by conducting Student t-test. A null hypothesis and an alternative hypothesis were proposed. The null hypothesis proposed states that there is no significant difference between the two groups and the alternative hypothesis states that there is a significant difference between the groups. The p-value for the test calculated as 0.0275 and found that the test is significant as the p-value obtained is less than 0.05. Hence the Null hypothesis was accepted (Table 5, Figure 3).
P. granatum peels used for the biosynthesis of silver nanoparticles showed inhibition against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli pathogens 23. Biosynthesized Ag NPs had reported as more effective than chemically synthesized Ag NPs.
P. granatum Ag NPs showed high zone of inhibition as compared to M. acuminata Ag NPs, which showed the least zone of inhibition against B. subtilis. The antimicrobial activity of chemically synthesised Ag NPs (9.2 ± 2.28 mm) was less efficient as compared to P. granatum Ag NPs (11.2 ± 2.68 mm), and it was more efficient as compared to M. acuminata Ag NPs (7.2 ± 3.63 mm) against B. subtilis. Chemically synthesized ZnO NPs (6.4 ± 3.29 mm) showed the least zone of inhibition as compared to M. acuminata ZnO NPs (10.4 ± 0.89 mm) and P. granatum ZnO NPs (14.8 ± 3.03 mm) whereas P. granatum ZnO NPs showed a higher zone of inhibition as compared to M. acuminata ZnO NPs. Biosynthesized ZnO NPs are more efficient as an antimicrobial agent as compared to biosynthesized Ag NPs against B. subtilis. Hence it is concluded that biosynthesized NPs are more efficient as compared to chemically synthesized NPs against B. subtilis.
The antimicrobial properties of chemically and biologically synthesized silver and zinc oxide against B. subtilis had analyzed conducting Student t-test. A null hypothesis and an alternative hypothesis had proposed. The null hypothesis proposed states that there is no significant difference between the two groups and the alternative hypothesis states that there is a significant difference between the groups. The p-value for the test calculated as 0.327 and found that the test is non-significant as the p-value obtained is more than 0.05. Hence the alternative hypothesis was accepted (Table 6, Figure 4).
The use of Musa peels for the synthesis of ZnO NPs were compared with commercial bulk ZnO particles and also with ZnO NPs prepared using nonionic and anionic surfactants24. It was found that the ZnO NPs films prepared from the Musa peels exhibited DPPH scavenging activity and was higher than that of the commercial film. Apart from this, the films with ZnO NPs exhibited antibacterial properties towards S. aureus and E. coli. chitosan/zinc oxide nanocomposites were tested against Gram-positive and Gram-negative bacteria and the results showed that the nanocomposite possesses thermal and antibacterial properties that got improved with the presence of ZnO NPs when compared with chitosan alone25.
Comparative analysis of antimicrobial activities of biosynthesized Ag NPs and ZnO NPs showed that biosynthesized Ag NPs were more efficient against E. coli as compared to biosynthesized ZnO NPs. Similarly biosynthesized ZnO NPs were more efficient against B. subtilis as compared to biosynthesized Ag NPs.