B6768

The Ag NFs were synthesized in three steps. Firstly, aqueous solutions of silver nitrate (High purity chemicals, 3692741, 0.3 M, 50 ml) and ammonium citrate dibasic (Sigma Aldrich, 09833, 0.15 M, 50 ml) were mixed for 2 minutes at 50 °C resulting in whitish precipitate of silver citrate complex22 (link). In the second step, an aqueous ammonia solution (Samchun Chemicals, A0628, 14.8 M) was added drop by drop until the solution became colorless, and an aqueous boric acid solution was added (Sigma Aldrich, B6768, 0.5 M, 50 ml)17 43 . Finally, an aqueous L-ascorbic acid solution (Sigma Aldrich, A5960, 0.1~1 M, 50 ml) was added15 18 25 . The color of the solution immediately turned into dark brown indicating synthesis of Ag NFs. After completing the reaction in 15 minutes, Ag NFs (~1.6 g) were collected by vacuum filtration (PTFE membrane, 0.2 μm) and repeatedly rinsed with deionized water. Ag NFs were then dried in an oven at 40 °C for 12 h. The synthesis could be upscaled by increasing the amounts of reagents.

Parallel cultures of B. subtilis (lab strain) were cultured in rich or mineral media in baffled flasks. Flasks were incubated at 37 °C with continuous stirring at 200 rpm. The rich media was prepared from 10 g/L tryptone (T9410, Sigma-Aldrich), 10 g/L NaCl (27810.295, VWR) and 5 g/L yeast extract (92144, Sigma-Aldrich). The mineral media was prepared in MilliQ-H₂O (MQ-H₂O) by dissolving 11.2 g/L Na₂HPO₄-7H₂O (S9390, Sigma-Aldrich), 3 g/L KH₂PO₄ (P5655, Sigma-Aldrich), 0.5 g/L NaCl (27810.295, VWR), 0.5 g/L NH₄Cl (A9434, Sigma-Aldrich), 0.2465 g/L MgSO₄-7H₂O (M5921, Sigma-Aldrich), 0.1470 g/L CaCl2-2H₂O (223506, Sigma-Aldrich), 4 g/L glucose (101176K, VWR) and 1 mL/L media of a trace element solution containing 10 g/L FeSO₄-7H₂O (F8633, Sigma-Aldrich), 2.25 g/L ZnSO₄-7H₂O (Z0251, Sigma-Aldrich), 2 g/L CaCl₂-2H₂O (223506, Sigma-Aldrich), 1 g/L CuSO₄-5H₂O (197722500, Thermo Fisher Scientific), 0.38 g/L MnCl₂-4H₂O (M5005, Sigma-Aldrich), 0.14 g/L H₂BO₃ (B6768, Sigma-Aldrich), and 0.1 g/L (NH₄)₆Mo₇O₂₄-4H₂O (1011820250, Merck Millipore, Damstadt, Germany). The final media was supplemented with 600 µg/L CoCl₂-6H₂O (33606, VWR), 1 mg/L biotin (47868, Sigma-Aldrich), and 1 mg/L thiamine hydrochloride (T1270, Sigma-Aldrich).

The glucose mineral medium for shake flasks was prepared in milliQ-H₂O (18.2 MΩ cm) by dissolving 11.2 g/l Na₂HPO₄–7H₂O (S9390, Sigma-Aldrich), 3 g/l KH₂PO₄ (P5655, Sigma-Aldrich), 0.5 g/l NaCl (27810.295, VWR), 1 g/l NH₄Cl (A9434, Sigma-Aldrich), 0.2465 g/l MgSO₄–7H₂O (M5921, Sigma-Aldrich), 4 g/l glucose (101176 K, VWR), 2 ml/l of a 50 mg/l CoCl₂–6H₂O solution (C8661, Sigma-Aldrich) and 2 ml/l medium of a trace element solution containing 10 g/l FeSO₄–7H₂O (F8633, Sigma-Aldrich), 2.25 g/l ZnSO₄–7H₂O (Z0251, Sigma-Aldrich), 2 g/l CaCl₂–2H₂O (223506, Sigma-Aldrich), 1 g/l CuSO₄–5H₂O (197722500, Fisher Scientific), 0.38 g/l MnCl₂–4H₂O (M5005, Sigma-Aldrich), 0.14 g/l H₂BO₃ (B6768, Sigma-Aldrich) and 0.1 g/l (NH₄)6Mo₇O₂₄–4H₂O (1011820250, Merck).