## 4 Discussion  
The novelty of this work was to use microwaves and fungal biomass to synthesize bi-metallic Ag-Cu NPs and optimize the process conditions using statistical methods. The synthesis process depended on the amount of fungal biomass used, the concentration of metals, irradiation power, and the reaction time, which we optimized for the final synthesis.  
The synthesized bi-metallic NPs showed antimicrobial activities against three clinically relevant Gram-negative bacterial pathogens, namely _K. pneumoniae, E. colaeae_, and _P. aeruginosa_. Three of the bacteria, Gram-negative _E. coli_ and Gram-positive _E. faecalis_ and _S. aureus_ were inhibited slightly. The higher inhibition of Gram-positive bacteria has been proposed, and for instance, _E. coli_ and _P. aeruginosa_ were inhibited less than _S. aureus_ by Zn and Cu NPs [36; 37]. Our study does not support the separation between Gram-positive and Gram-negative bacteria. It may be that the NP characteristics may be of great importance, in addition to the resistance of the particular bacterial strain [38]. Ag NPs have been shown to efficiently inhibit _P. aeruginosa_, _E. coli_, and _S. aureus_. _E. coli_ was inhibited, for instance, by Zn NPs [39] and Ag NPs [40]. Moreover, methicillin-resistant _S. aureus_ (MRSA) has recently been shown to be inhibited by NPs [41; 42; 43]. For some reason, our bi-metallic NPs did not inhibit _S. aureus_ especially efficiently. More than double the minimum inhibitory concentration and thrice the minimum bactericidal concentration were needed to inhibit _S. aureus_ compared to _P. aeruginosa_. All bacterial species that we tested have been inhibited by some metal NPs previously, at least to some extent [44; 45; 46; 47; 48].  
Bi-metallic NPs are promising antimicrobial agents, especially against multidrugment pathogenic microorganisms [49]. The bacteria _S. pneumoniae, P. aeruginosa, E. coli_, _Bacillus subtilis_, and _S. aureus_ were inhibited efficiently by Ag-Cu NPs previously [50; 51; 52]. The ultra-high antibacterial activity of 2 nm size Ag-Cu NPs [53] was observed against  
Figure 10: Cytotoxicity at different concentrations of Ag-Cu NPs (mean and SD, n = 3) after 24 and 48 h.  
E. coli_ and _S. aureus_. Ag-Cu NPs have been proposed to be useful to inhibit bacteria in food packages [54; 55; 56].  
In general, NPs with a size of 1-100 nm have shown strong antimicrobial effects against both Gram-positive and Gram-negative bacteria [57]. Smaller particles are more efficient because the ratio of the surface region to the volume increases when the particle size diminishes. Our NPs were relatively small, varying between 12 and 30 nm. Although only the dynamic light scattering ((DLS) method would give a precise size of the NPs, we suggest that the process we propose here is suitable for producing small and efficient bi-metallic NPs.  
Microwave power and optimization of the synthesizing conditions using RSM have been carried out only recently. By optimizing the process, very small Se and Cu nanoparticles (1-10 nm) were synthesized using a microwave power of 790 W for 15 min [58; 59]. In our tests, the highest power, 500 W, was not the most efficient in producing bi-metallic Ag-Cu NPs. Instead, the highest yield was reached in 15 min with 350 W. Elsewhere, a constant 800 W power, and the maximum tested time (6 min) produced Ag NPs of 46 nm size [60]. Relatively small Fe NPs (14 nm) were produced in 10 min [61]. We were able to synthesize smaller than 30 nm bi-metallic NPs. It is possible that testing a larger scale of parameter values would produce smaller NPs, which is a future challenge.