# J524: METHANE OXIDIZING BACTERIUM MEDIUM


## Main sol. J524

30	g	NaCl	
0.14	g	KH2PO4	
0.8	g	CaCl2 x 2 H2O	
3.4	g	MgSO4 x 7 H2O	
4.2	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.25	g	NaNO3	
0.25	g	NH4Cl	
0.2	g	Na2SiO3 x 9 H2O	
20	mg	Fe2(SO4)3 x n H2O	
0.5	mg	Na2SeO3 x 5 H2O	
0.5	mg	NiCl2 x 6 H2O	
3	ml	**Trace mineral solution**	
1000	ml	Distilled water	

1. Mix components, adjust pH to 6.0 - 6.5 and autoclave. After cooling, add the following solutions (filter-sterilized) to the medium: 

1	ml	**Trace vitamins**	
10	ml	NaHCO3 (5%)	
2	ml	CuSO4 (1 mM)	

2. Distribute the medium into culture vessels (leave >80% volume gas phase) under a N2-CO2 (4:1) gas mixture and seal with butyl rubber stoppers. Pressurize the culture vessels to 200 kPa with methane gas. Finally add O2 gas to make up 2% O2 concentration in the gas phase. Readjust pH to 6.0 - 6.3, if necessary.


## Trace mineral solution

1.5	g	Nitrilotriacetic acid	
0.5	g	MnSO4 x n H2O	
0.18	g	CoSO4 x 7 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	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. Then proceed to add minerals. Adjust final pH to 7.0.


## 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