# J794: METHANOBACTERIUM MEDIUM (III) ## Main sol. J794 0.136 g KH2PO4 0.54 g NH4Cl 0.2 g MgCl2 x 6 H2O 0.147 g CaCl2 x 2 H2O 2.5 g NaHCO3 0.2 g Yeast extract 0.8 g Sodium acetate 10 ml **Trace vitamins** 10 ml **Trace minerals** 1 mg Resazurin 1000 ml Distilled water 1. Mix components, bring to a boil for several seconds and cool down under a stream of H2-CO2 (80:20, v/v). Distribute the medium into culture vessels (e.g., 5 ml in the Hungate tube) under the same gas mixture, seal with butyl rubber stoppers and autoclave. Add per liter the following sterile solutions from anaerobic stocks (autoclaved under a N2 atmosphere): 10 ml L-Cysteine HCl x H2O (5%) 10 ml Na2S x 9 H2O (5%) 2. Pressurize the inoculated vessels to 150 kPa H2-CO2 (80:20, v/v). ## Trace vitamins 2 mg Biotin 2 mg Folic acid 10 mg Pyridoxine hydrochloride 5 mg Thiamine HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium pantothenate 0.1 mg Vitamin B12 5 mg p-Aminobenzoic acid 5 mg Lipoic acid 1000 ml Distilled water ## Trace minerals 1.5 g Nitrilotriacetic acid 3 g MgSO4 x 7 H2O 0.5 g MnSO4 x n H2O 1 g NaCl 0.1 g FeSO4 x 7 H2O 0.1 g CoSO4 x 7 H2O 0.1 g CaCl2 x 2 H2O 0.1 g ZnSO4 x 7 H2O 0.01 g CuSO4 x 5 H2O 0.01 g AlK(SO4)2 0.01 g H3BO3 0.01 g Na2MoO4 x 2 H2O 1000 ml Distilled water 1. Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0.