# 1131: HYDROGENIVIRGA OKINAWENSIS MEDIUM


## Main sol. 1131

1000	ml	**Modified MJ synthetic seawater**	

1. Sparge medium with 100% N2 gas mixture for 10 min to make anoxic.
2. Adjust pH to 7.0 with NaOH.
3. Sparge medium with 80% N2 and 20% CO2 gas mixture for 10 min.

3	g/l	Sulfur (powder)	

4. Dispense the medium (10 mL) into 100 mL serum bottles, containing 0,03g sulfur (powder) under a stream of H2/CO2 (80:20) and seal with butyl rubber stoppers.
5. Autoclave at 121 °C for 20 min.
6. Prior to inoculation, add vitamins, NaNO3, and NaHCO3 (from filter sterilized stock solutions under a N2 gas atmosphere) to the medium.

10	ml/l	**Vitamin solution**	
1	g/l	NaNO3	
1	g/l	NaHCO3	


## Modified MJ synthetic seawater

30	g	NaCl	
0.14	g	K2HPO4	
0.5	g	CaCl2 x 2 H2O	
0.5	g	NH4Cl	
3.4	g	MgSO4 x 7 H2O	
4.18	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.01	g	FeSO4 x 7 H2O	
1	mg	NiCl2 x 6 H2O	
1	mg	Na2SeO3 x 5 H2O	
10	ml	**Modified Wolin's mineral solution II**	
1000	ml	Distilled water	


## Vitamin solution

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine-HCl x 2 H2O	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	D-Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water	


## Modified Wolin's mineral solution II

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.025	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.