Filter sterilized methanol

The artificial seawater (ASW) medium was composed of (for 1 liter solution): 27.5 g NaCl, 10.68 g MgCl₂*6H₂O, 2 g MgSO₄*7H₂O, 1 g KCl, 0.5 g CaCl₂, 456 µL of FeSO₄*7H₂O 4 g/L, five mL of KH₂PO₄51.2 g/L, five mL of Na₂HPO₄ 34 g/L. The Instant Ocean (IO) seawater medium was bought from Aquarium systems (Mentor, OH, USA) and dissolved at 30 g/L. Estimation of its composition and comparison with the ASW medium is provided in Table S1. Both media were supplemented with one mL of trace elements (FeSO₄*7H₂O 0.9 g/L, CuSO₄*5H₂O 0.03 g/L and MnSO₄*H₂O 0.2 g/L) and with NaNO₃ (Fisher Scientific Canada, Ottawa, ON, Canada) at various concentrations, depending on the experiment (Tables 1 and 2). The pH was adjusted (NaOH) at 8.0 before autoclaving. Filter-sterilized methanol (Fisher Scientific) was then added as a carbon source to support bacterial growth, also at various concentrations depending on the experiment (Tables 1 and 2). The sterile media were distributed (60 ml) in sterile serologic vials, which included twenty carriers (Bioflow 9 mm; Rauschert, Steinwiessen, Allemagne). Prior to use, these carriers were washed with HCl 10% (v/v) for 3 h, rinsed with water and autoclaved. The vials were purged of oxygen for 10 min with nitrogen gas (Praxair, Mississauga, ON, Canada) and sealed with sterile septum caps.

We used Hypho minimal medium [20 ] with the following composition. P solution: dipotassium phosphate (K₂HPO₄.7H₂O) 33.1g/L and sodium dihydrogen phosphate (NaH₂PO₄.H2O) 25.9g/L; S solution: ammonium sulphate ((NH4)₂.SO₄) 5g/L and magnesium sulphate (MgSO₄.7H₂O) 2 g/L (Fisher Scientific); metal mix 1mL/L, pH 7). As appropriate, we supplemented it with 120mM filter-sterilized methanol (Fisher Scientific) as the sole carbon source, or other carbon sources (described below). We incubated cultures at 30°C for 48h (liquid; with shaking) or 5 days (agar plates), unless mentioned otherwise. For long-term storage, we made glycerol stocks by mixing 500μL of liquid culture with 500μL of 50% glycerol (Merck) (in sterile water), and stored at -80°C.