# J872: METHANOBACTERIUM MEDIUM (IV)


## Main sol. J872

0.5	g	KH2PO4	
0.4	g	MgSO4 x 7 H2O	
5	g	NaCl	
0.4	g	NH4Cl	
0.05	g	CaCl2 x 2 H2O	
2	mg	FeSO4 x 7 H2O	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
1	g	Yeast extract	
50	ml	**Sludge fluid**	
1	g	Sodium acetate	
2	g	Sodium formate	
4	g	NaHCO3	
20	ml	**Fatty acid mixture**	
1	mg	Resazurin	
930	ml	Distilled water	

1. Mix components except NaHCO3 and bring to a boil for 5-10 sec. Cool down under a stream of H2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense the medium into culture vessels under a stream of H2-CO2 (80:20, v/v), seal with butyl rubber stoppers and autoclave. Prior to inoculation, add per liter the following solutions (autoclaved and stored under a N2 atmosphere): 

10	ml	Na2S x 9 H2O (5%)	
10	ml	L-Cysteine HCl x H2O (5%)	

2. Pressurize inoculated vessels to 200 kPa with a H2-CO2 (80:20, v/v) gas mixture.


## Sludge fluid

4	g	Yeast extract	
1000	ml	Sludge	

1. Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark.


## FeCl2 solution

10	ml	HCl (7.7 M, 25%)	
1.5	g	FeCl2 x 4 H2O	
990	ml	Distilled water	


## Trace element solution

70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	


## Fatty acid mixture

0.5	g	Valeric acid	
0.5	g	Isovaleric acid	
0.5	g	alpha-Methylbutyric acid	
0.5	g	Isobutyric acid	
20	ml	Distilled water	

1. Adjust pH to 7.5 with concentrated NaOH solution.