Biosynthesis of Silver Nanoparticles

The synthesis methodology of AgNPs was prepared according to [20 (link),21 (link),22 (link)]. The tested Geobacillus strains were grown aerobically in 100 mL liquid broth with the following composition (in g/L): tryptone (Roth, Karlsruhe, Germany), 10; meat extract (Merck, Rahway, NJ, USA), 5; NaCl (Merck), 5; CaCl₂ (Merck), 2.3 mM; and ZnSO₄ (Merck), 0.91 µM, in 250 mL Erlenmeyer flasks. The cultures were grown in an orbital shaker (Esco, Singapore, Singapore) at 55 °C with aeration at 180 rpm. After 48 h of incubation, cells were separated by centrifugation at 16,000× g for 10 min. Cell-free secretomes were used as material for AgNP synthesis. The secretomes of the target strains were treated with AgNO₃ (Roth) solution at final concentrations of 2 mM. The whole mixtures were incubated in a shaking incubator for 48 h at 55 °C and 200 rpm. The secretomes without AgNO₃ and bacterial growth medium supplemented with 2 mM AgNO₃ were used as controls. After 48 h of incubation, the mixtures were centrifuged at 3000× g for 10 min to remove media components. Then, mixtures were centrifuged at 16,000× g for 15 min to collect AgNPs. To remove unconverted silver ions, the obtained pellets were washed three times with 70% ethanol and three times with deionized water by centrifugation at 16,000× g for 15 min.