## 3 Results and Discussion
### Analyses of Size Distribution and Zeta Potential  
The average particle size and zeta potential values obtained from all samples (with adjusted pH values before and after AuNP preparation) are shown in Figures 14 and 15. The data (Figure 15) show that all AuNPs exhibited negative zeta potential (in all pH variants). However, at alkaline pH, the AuNPs held a more negative charge than at acidic pH. Moreover, more favorable zeta potential was obtained in the samples that had a pH that was adjusted after AuNP preparation. Acidic pH induced the production of larger AuNPs with unfavorably low negative charge, which promoted agglomeration. Figure 14 shows the hydrodynamic diameter (Z-average) at different pH values. The size distribution analysis (Figure 14) confirmed that adjusting the pH after AuNP production resulted in better peaks. It was reported previously that an alkaline environment is preferred in the biological production of nanoparticles [37], because the repulsion between H\({}^{+}\) and Au\({}^{+}\) ions in the acidic environment decreases the chance of assembling Au\({}^{+}\) ions into AuNPs. In this study, we report, for the first time, that acidic pH is unfavorable for the biological production of AuNPs due to the nearly neutral zeta potential of the produced nanoparticles. The zeta potential being close to zero resulted in unstable nanoparticles with a tendency to agglomerate. Since the preferred nanoparticle size for medical application is below 50 nm [38], the AuNPs produced or placed in the acidic pH range are suboptimal due to the fact of their larger size. Thus, to obtain AuNPs of preferable size and zeta potential by the biological method, we propose to prepare them with an initial pH of 10. Subsequent lowering of the environmental pH to the physiological pH (close to 7.2) results in AuNPs with favorable size and zeta potential. Figure 13: SEM and EDS images obtained from different samples: (**A,B**) SEM results of the washed sample at pH 10; (**C**) EDS results of the washed sample at pH 10. The samples were placed on glow-discharge-activated silicon wafers. [PERSON] et al. used _Penicillium rugulosum_ for the preparation of AuNPs and adjusted the mycelium-free supernatant to different pH values of 2, 4, 6, 8, and 10 and showed that the best pH values were between four and six [39], which is different from this study, where the best pH values were between 6 and 10. In addition, [PERSON] et al. showed that the best pH values for the production of AuNPs by _Saccharomyces cerevisiae_ were between pH four and six [40], and [PERSON] et al. showed that the best pH values for the production of AuNPs by _Penicillium bevacizumab_ were between five and eight [41]. These differences may be due to the differences in the capping agents that were secreted by the fungal strains. In addition, they did not use other methods to analyze the size distribution, such as DLS or SEM, and  
Figure 14: Average size (hydrodynamic diameter) of AuNPs obtained at different pH values. Figure 15: Zeta potential of the obtained AuNPs at different pH values. The different pH values were adjusted before and after the preparation of AuNPs. they did not compare the effects of different pH values before and after the production of AuNPs on the nanoparticle size distribution, zeta potential, and type of capping agent. Unlike the other methods of AuNP production (i.e., chemical and physical methods), which offer the easy production of positively charged nanoparticles [42; 43], this cannot be achieved using the biological production method. In chemical or physical methods of nanoparticle production, the charge of the nanoparticles depends on the charge of the reducing and stabilizing agent [2]. In the biological method of nanoparticle production, the charge depends on capping agents, which are reported to be secreted microbial proteins. There are some reports that use SDS-PAGE and FTIR techniques to reveal those proteins [25; 44]. In 2010, [PERSON] et al.