368. METHANOBACTERIUM VETERUM MEDIUM

Solution 1:

Na-acetate × 3 H₂O 0.5 g

(NH₄)₂SO₄ 0.45 g

K₂HPO₄ 0.29 g

KH₂PO₄ 0.18 g

MgSO₄ × 7 H₂O 0.12g

CaCl₂ × 2 H₂O 0.06 g

NaCl 0.05

Resazurin 0.01 g

Na₂CO₃ 5.0 g

Trace element solution (see below) 10.0 ml

Vitamin solution (see below) 10.0 ml

Distilled water 965.0 ml

Solution 2 (reducing agents):

L-Cysteine-HCl × H₂O 0.5 g

Na₂S × 9 H₂O 0. 5 g

Distilled water 10.0 ml

Trace element solution:

Nitrilotriacetic acid 1.5 g

MgSO₄ × 7 H₂O 3.0 g

MnSO₄ × 7 H₂O 0.5 g

NaCl 1.0 g

FeSO₄ × 7 H₂O 0.1 g

CoSO₄ 0.18 g

CaCl₂ × 2 H₂O 0.1 g

ZnSO₄ 0.18 g

CuSO₄ 0.01 g

KAl(SO₄)₂ × 12 H₂O 0.02 g

H₃BO₃ 0.01 g

Na₂MoO₄ × 2 H₂O 0.01 g

NiCl₂ 0.025 g

Na₂SeO₃ × 5 H₂O 0.3 mg

Distilled water 1000.0 ml

pH 7.0-7.4

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine 10.0 mg

Riboflavin 5.0 mg

Pantotenoic acid 5.0 mg

p-Aminobenzoic acid 5.0 mg

Thiamine-HCl 5.0 mg

Nicotinic acid 5.0 mg

Vitamin B₁₂ 0.1 mg

Lipoic (thioctic) acid 5.0 mg

Distilled water 1000.0 ml

pH 7.2-7.4

Prepare medium in anaerobic conditions, blowing through with N₂ without O₂ up to sterilization. Solutions of reducing agents (10.0 ml) and others solutions add to base medium after separate sterilization at 121°C for 15 min. Sterilize vitamin solution by filtration.

Cultivate in a gas mixture of 80% H₂ and 20% CO₂.

 

Updated 10.08.2021