NaNO3 0.20 g KH2PO4 0.20 g NH4Cl 0.20 g MgCl2 x 6 H2O 0.40 g KCl 0.20 g CaCl2 x 2 H2O 0.10 g Trace element solution SL-4 2.00 ml Na2S2O3 x 5 H2O 2.50 g Distilled water 1000.00 ml 1. Dissolve ingredients, except sodium thiosulfate, then sparge solution with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 7.0, then distribute medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials (e.g., 20 ml medium in 100 ml bottles) and autoclave. Prior to inoculation, add thiosulfate from an anoxic stock solution prepared under 100% N2 gas and sterilized by filtration. Adjust pH of complete medium to 7.0, if necessary. 2. After inoculation, pressurize culture vessels to 0.5 bar overpressure with sterile 100% H2 gas. Trace element solution SL-4 (from medium 14) Na2-EDTA 0.50 g FeSO4 x 7 H2O 0.20 g ZnSO4 x 7 H2O 0.10 g MnCl2 x 4 H2O 0.03 g H3BO3 0.30 g CoCl2 x 6 H2O 0.20 g CuCl2 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.02 g Na2MoO4 x 2 H2O 0.03 g Distilled water 1000.00 ml First dissolve EDTA in distilled water and adjust pH to 7.0 using 2 N NaOH; then add other compounds.
