M5921

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).