# J403: ACETOBACTERIUM SUBMARINUS MEDIUM


## Main sol. J403

30	g	Sea Salt (Sigma)	
0.1	g	Yeast extract (BD-Difco)	
1	ml	**Trace element solution**	
1	ml	**Vitamin solution**	
1	ml	**Thiamine solution**	
1	ml	**Vitamin B12 solution 0.05 g/L**	
30	mg	Na2WO4 x 2 H2O	
0.5	mg	Na2SeO4	
1	g	Sodium acetate	
2	g	Methylamine	
5	g	Sodium formate	
5	ml	Methanol	
0.2	g	KH2PO4	
0.27	g	NH4Cl	
0.5	mg	Resazurin	
1.26	g	NaHCO3	
0.5	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Mix ingredients except methanol, NaHCO3 and Na2S x 9H2O, bring to a boil and cool down under a stream of N2 gas. Add methanol to the medium and distribute into culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-CO2 (4:1, v/v) gas mixture, seal with butyl rubber stoppers, and autoclave. Separately autoclave 5% Na2S x 9H2O solution under a N2 atmosphere. Filer-sterilize 8% NaHCO3 solution and store under a N2-CO2 (4:1, v/v) gas mixture. Before inoculation, add the sterile NaHCO3 and Na2S x 9H2O solutions to the medium, and readjust pH to 7.0, if necessary. Pressurize the inoculated bottles to 200 kPa H2-CO2 (4:1, v/v).


## Vitamin solution

4	mg	p-Aminobenzoic acid	
1	mg	Biotin	
10	mg	Nicotinic acid	
5	mg	DL-Calcium pantothenate	
10	mg	Pyridoxine hydrochloride	
100	ml	Sodium phosphate buffer (10 mM, pH 7.1)	


## Thiamine solution

10	mg	Thiamine HCl	
100	ml	Sodium phosphate buffer (25 mM, pH 3.4)	


## Vitamin B12 solution 0.05 g/L

5	mg	Vitamin B12	
100	ml	Distilled water	


## Trace element solution

12.8	g	Nitrilotriacetic acid	
0.42	g	FeSO4 x 7 H2O	
0.01	g	H3BO3	
0.1	g	MnCl2 x 4 H2O	
0.17	g	CoCl2 x 6 H2O	
0.02	g	CuCl2 x 2 H2O	
0.21	g	ZnSO4 x 7 H2O	
0.01	g	Na2MoO4 x 2 H2O	

1. Dissolve nitrilotriacetic acid in 800 ml distilled water and adjust pH to 6.5 with NaOH. Then proceed to add the minerals and bring volume to 1.0 L.