# J928: METHYLOBACTER MR1 MEDIUM


## Main sol. J928

20	g	NaCl	
0.09	g	KH2PO4	
0.26	g	Na2HPO4	
0.5	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	
0.02	g	FeSO4 x 7 H2O	
3	ml	**Trace mineral solution**	
0.1	ml	CuSO4 x 5 H2O (1 mM)	
1000	ml	Distilled water	

1. Mix components, adjust pH to 6.6 - 6.8 and autoclave. After cooling, add the following solutions (filter-sterilized) to the medium: 

1	ml	**Trace vitamins**	
1.25	ml	NaHCO3 (8%)	

2. Distribute the medium into culture vessels (leave >80% volume gas phase), purge air in the gas phase with N2 for 1 min and seal with butyl rubber stoppers. Inject O2 gas into the gas phase (1.5% volume of the gas phase). Then, overpressure the culture vessels with methane gas (50% volume of the gas phase).


## Trace mineral solution

1.5	g	Nitrilotriacetic acid	
0.5	g	MnSO4 x n H2O	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.03	g	Na2MoO4 x 2 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.02	g	Na2WO4 x 2 H2O	
0.01	g	NiCl2 x 6 H2O	
5	mg	Na2SeO3 x 5 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