Znso4

U937-PR9 and NB4 cells were cultured in RPMI 1640 (Thermo Fisher, A10491), supplemented with 10% fetal bovine serum (Thermo Fisher, 10082147), and incubated at 37 °C, 5% CO₂. For the zinc induction of U937-PR9 cells, ZnSO₄ (Sigma, Z0251) was dissolved in sterile water as a stock solution at 100 mM. ZnSO₄ was added directly to the culture medium at a final concentration of 100 μM and incubated at 37 °C for 5 h, as described previously^{15 (link)}. For the non-induced U937-PR9 cells, sterile water was added instead of ZnSO₄ and incubated under the same conditions.

Stage 7 medium was based on a previous report [7 (link)] with our original modifications. Cells were cultured in the MCDB 131 medium with 1% P/S, 2% fat-free bovine serum albumin (FUJIFILM Wako), 20 mM glucose, 1.5 g/L NaHCO₃, 1% GlutaMAX, 0.5% ITS-X (Thermo Fisher Scientific), 10 μM ALK5iII or the alternative candidates, 1 μM T₃, 10 μM ZnSO₄ (Merck Millipore), 1.4 IU/mL heparin sodium salt (Nacalai Tesque, Kyoto, Japan), 1 mM N-acetyl cysteine (Merck Millipore), 10 μM Trolox (FUJIFILM Wako), 2 μM R428 (Selleck Chemicals, Houston, TX, USA), 1 μM PD-166866, 3 μM TR06141363 (Multi-kinase inhibitor, Takeda Pharmaceutical Company), and 10 μM Y-27632, for 4 days. To generate iPICs for implantation, cells were dissociated and re-sized in an Elplasia 6-well microwell plate (Corning Incorporated, Corning, NY, USA) or a gas-permeable microwell culture bag (Toyo Seikan Group Holdings, Ltd., Yokohama, Japan) [20 (link)] and cultured in the stage 7 medium.

ZnS nanoparticles were synthesized by delivering a H₂S gas stream produced by a sulfidogenic bioreactor to an aqueous solution of 5 mM ZnSO₄ (Merck Millipore, Burlington, MA, USA). A 2.3 L working volume sulfidogenic bioreactor (Fermac 200; Electrolab Biotech, Tewkesbury, United Kingdom) was operated as an upflow biofilm continuous sulfidogenic bioreactor at pH 7.0, as described elsewhere [24 (link)], housing sulfidogenic bacteria dominated by the SRB genera of Desulfomicrobium, Desulfobacterium and Desulfovibrio that were obtained from different ponds in Salar de Huasco, Chile [25 (link)] having blackened sediments. Enrichments were grown on porous beads of recycled glass of 1–2 mm diameter. The bioreactor was fed 5 mM lactate, 1000 ppm sulfate, autotrophic basal salt (ABS; [26 (link)]), trace elements and yeast extract at pH of 6.5. The H₂S gas was removed by a stream of oxygen-free nitrogen (OFN) at a flux to 100 mL·min⁻¹ and transferred to an off-line vessel containing 50 mL of an aqueous solution of ZnSO₄. After the zinc solution was sparged with gas for 60 min, the precipitate formed (ZnS nanoparticles) was collected from the solution, dried at 30 °C for 24 h and stored for further use.

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.