115. METHANOBACTERIUM MEDIUM

KH₂PO₄ 0.5 g

MgSO₄ × 7 H₂O 0.4 g

NaCl 0.4 g

NH₄Cl 0.4 g

CaCl₂ × 2 H₂O 0.05 g

Trace element solution SL-10 (see below) 1.0 ml

Vitamin solution (see below) 5.0 ml

Yeast extract 1.0 g

Na-acetate 1.0 g

Na-formate 2.0 g

NaHCO₃ 4.0 g

Resazurin 1.0 mg

L-Cysteine-HCl 0.5 g

Na₂S × 9 H₂O 0.5 g

Fatty acid mixture (see below) 20.0 ml

Sludge water: (see below) 50.0 ml

Distilled water 940.0 ml

Trace element solution SL-10:

HCl (25%; 7.7 M) 10.0 ml

FeCl₂ × 4 H₂O 1.5 g

ZnCl₂ 70.0 mg

MnCl₂ × 4 H₂O 100.0 mg

H₃BO₃ 6.0 mg

CoCl₂ × 6 H₂O 190.0 mg

CuCl₂ × 2 H₂O 2.0 mg

NiCl₂ × 6 H₂O 24.0 mg

Na₂MoO₄ × 2 H₂O 36.0 mg

Distilled water 990.0 ml

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine (B₂) 10.0 mg

Riboflavin (B₁) 5.0 mg

Pantotenoic acid 5.0 mg

p-Aminobenzoic acid 5.0 mg

Thiamine-HCl 5.0 mg

Nicotinic acid 5.0 mg

Cyanocobalamin (B₁₂) 0.1 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

Fatty acid mixture:

Valeric acid 0.5 g

Isovaleric acid 0.5 g

α-Methylbutyric acid 0.5 g

Isobutyric acid 0.5 g

Distilled water 20.0 ml

pH 7.5 (adjust with concentrated NaOH)

Sludge water: to sludge from an anaerobic digester add 0.4% yeast extract and after gassing with

N₂ gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13,000 g and autoclave the resulting clear supernatant under N₂ gas at 121°C for 15 min (pH 6.7-7.0). Base medium and trace element solution autoclave at 121°C for 15 min. Vitamin solution is filter sterilized. Prepare the medium anaerobically under a gas atmosphere of 80% H₂ and 20% CO₂.

 

Updated 1.02.2024