Solution A: NH4Cl 0.25 g NaH2PO4 0.60 g KCl 0.10 g NaHCO3 2.50 g NaClO3 1.00 g Na-Acetate 1.36 g Distilled water 1000.00 ml Solution B: MgSO4 30.00 mg CaCl2 x 2 H2O 10.00 mg Distilled water 10.00 ml Solution C: Na2MoO4 25.00 mg Na2WO4 x 2 H2O 25.00 mg Distilled water 1000.00 ml Solution D: Trace element solution SL-10 HCl (25%; 7.7 M) 10.00 ml FeCl2 x 4 H2O 1.50 g ZnCl2 70.00 mg MnCl2 x 4 H2O 100.00 mg H3BO3 6.00 mg CoCl2 x 6 H2O 190.00 mg CuCl2 x 2 H2O 2.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 990.00 ml First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.0 ml. Solution E: Vitamins Vitamin B12 50.00 mg Pantothenic acid 50.00 mg Riboflavin 50.00 mg Pyridoxamine-HCl 10.00 mg Biotin 20.00 mg Folic acid 20.00 mg Nicotinic acid 25.00 mg Nicotine amide 25.00 mg α-lipoic acid 50.00 mg p-aminobenzoic acid 50.00 mg Thiamine-HCl x 2 H2O 50.00 mg Distilled water 1000.00 ml
Stir for some hours, filter sterilize the solution. continued Autoclave solutions A, B and C separately and anaerobically under 80% N2 + 20% CO2 atmosphere. Combine 1000 ml solution A, 10 ml solution B and 10 ml solution C. Add 5 ml vitamin solution E and 1 ml trace element solution D. The pH should be 7.2 without further adjustment. Reactivate ampoules of DSM 13638 in medium 1 under aerobic conditions first, then transfer to anaerobic medium 908.
