# 1011c: DESULFOTHERMUS MJ MEDIUM (H2/CO2)


## Main sol. 1011c

30	g	NaCl	
0.14	g	K2HPO4	
0.14	g	CaCl2 x 2 H2O	
3.4	g	MgSO4 x 7 H2O	
4.18	g	MgCl2 x 6 H2O	
0.33	g	KCl	
0.25	g	NH4Cl	
0.01	g	Fe(NH4)2(SO4)2 x 6 H2O	
10	ml	**Modified Wolin's mineral solution**	
1.5	g	Na2CO3	
0.5	g	Na-pyruvate	
0.5	g	Na-lactate	
0.1	g	Yeast extract	
1.5	g	Na2S2O3 x 5 H2O	
1	ml	**Wolin's vitamin solution (10x)**	
1	ml	**Na-dithionite solution (5% w/v)**	
1000	ml	Distilled water	

1. Dissolve ingredients (except carbonate, pyruvate, lactate, yeast extract, thiosulfate, vitamins and dithionite), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials up to a volume of 20% and autoclave. Add pyruvate, lactate, yeast extract, thiosulfate and vitamins to the autoclaved medium from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate and dithionite from sterile anoxic stock solutions prepared under 80% N2 and 20% CO2 gas mixture. Solutions of vitamins, thiosulfate and dithionite are sterilized by filtration. Adjust pH of the complete medium to 6.7.
2. After inoculation pressurize vessels to 2 bar overpressure with sterile 80% H2 and 20% CO2 gas mixture.


## Modified Wolin's mineral solution

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	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 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.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## Na-dithionite solution (5% w/v)

50	g	NaHCO3	
50	g	Na2S2O4	
1000	ml	Distilled water	

1. Dissolve NaHCO3 in water and make the solution anoxic by sparging with 80% N2 and 20% CO2 gas mixture. Then dissolve the Na-dithionite and filter sterilize the solution into anoxic Hungate tubes. Store the prepared solution in the dark and refrigerated. Prepare only small amounts of stock solution, as Na-dithionite decomposes rapidly.