176. METHANOBACTERIUM MEDIUM

Solution 1:

NaCl 0.9 g

MgCl2 7 H2O 0.2 g

CaCl2 2 H2O 0.1 g

NH4Cl 1.0 g

Yeast extract 2.0 g

Resazurin 0.01 g

Trace element solution (see below) 10.0 ml

Vitamin solution (see below) 5.0 ml

Buffer solution a (see below) 10.0 ml

Buffer solution b (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

Buffer solutions:

a) K2HPO4 29.0 g

Distilled water 100.0 ml

b) KH2PO4 15.0 g

Distilled water 100.0 ml

Trace element solution:

Nitrilotriacetic acid 12.8 mg

FeSO4 7 H2O 0.1 mg

MnCl2 6 H2O 0.1 mg

CoCl2 2 H2O 0.17 mg

CaCl2 2 H2O 0.1 mg

ZnCl2 0.1 mg

CuCl2 2 H2O 0.02 mg

H3BO3 0.01 mg

Na2MoO4 2 H2O 0.01 mg

NaCl 1.0 mg

Na2SeO4 0.017 mg

Distilled water 1000.0 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

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 autoclaving at 121C for 15 min. Sterilize vitamin solution by filtration.

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

 

Updated 28/05/2020