Prepare the Thauer's vitamin solution and trace element solutions according to the recipes below. Aliquot for future use.

Dissolve in 900 ml demineralized water:

A	B
NaCl	22
MgCl2 x 6 H2O	4.5
MgSO4 x 7 H2O	4.5
CaCl2 x 2 H2O	0.675

Autoclave at 121°C for 20 min at 15 psi.

After cooling to room temperature add (in the flow cabinet) from sterile stocks:

A	B
1M KH2PO4	0.261
TES I	0.45
TES II	0.9
Thauer’s vitamin solution	0.45
NaHCO3	1.8

Adjust the pH to 7.8 using sterile 1M NaHCO3 solution.

Dissolve in 1l demineralized water:

A	B
Biotin	0.02
Folic Acid	0.02
Pyridoxine HCl	0.1
Thiamine HCl	0.05
Riboflavin	0.05
Nicotinic Acid	0.05
DL Pantothenic Acid	0.05
p Aminobenzoic Acid	0.05
Choline Chloride	2
Vitamin B12	0.01

Adjust to pH 7.0 with KOH.

Filter sterilize (0.2 µm) and store at 4ºC.

Dissolve 10 g NTA (Trisodium Nitrilotriacetate) in 500 ml demineralized water.

Adjust to pH 8 using NaOH pellets.

Add 5 g FeSO4 x 7 H2O and dissolve.

Top up with demineralized water to a total volume of 1l.

Filter sterilize (0.2 µm) or autoclave at 121°C for 20 min at 15 psi.

Store in the dark (slightly light sensitive) at RT.

Dissolve 5.5 g NTA (Trisodium Nitrilotriacetate) in 500 ml demineralized water.

Adjust to pH 8 using NaOH pellets.

Add the following ingredients and dissolve:

A	B
ZnSO4 x 7 H2O	0.43
CoCl2 x 6 H2O	0.24
MnCl2 x 4 H2O	0.99
CuSO4 x 5 H2O	0.25
Na2MoO4 x 2 H2O	0.22
NiCl2 x 6 H2O	0.19
NaSeO4	0.1075
H3BO3	0.14
CeCl3 x 7 H2O	0.24

Top up with demineralized water to a total volume of 1l.

Filter sterilize (0.2 µm) or autoclave at 121°C for 20 min at 15 psi.

Store in the dark (slightly light sensitive) at RT.