1011c .
DESULFOTHERMUS MJ MEDIUM (H2/CO2)
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* Medium for Sulfurovum aggregans DSM 27205
Supplement medium with 1.00 g/l NaNO3 and omit pyruvate, lactate, yeast extract and dithionite. Upon autoclaving add 10.00 g/l sterile sulfur powder (sterilized by steaming for 3 hours on each of 3 successive days) and adjust pH to 6.0 - 6.5. After inoculation do not pressurize with 80% H2 and 20% CO2 gas mixture and add sterile air in an amount that is equivalent to a volume of 10% of the headspace.
Growth conditions: 2-3 days
microaerobic
32 °C
Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
* 6.0 - 6.5
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 30.00 | g | ||
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K2HPO4 | 0.14 | g | ||
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CaCl2 x 2 H2O | 0.14 | g | ||
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MgSO4 x 7 H2O | 3.40 | g | ||
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MgCl2 x 6 H2O | 4.18 | g | ||
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KCl | 0.33 | g | ||
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NH4Cl | 0.25 | g | ||
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Fe(NH4)2(SO4)2 x 6 H2O | 0.01 | g | ||
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Modified Wolin's mineral solution | 10.00 | ml | ||
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Na2CO3 | 1.50 | g | ||
| Na-pyruvate* | 0.50 | g | |||
| Na-lactate* | 0.50 | g | |||
| Yeast extract* | 0.10 | g | |||
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Na2S2O3 x 5 H2O | 1.50 | g | ||
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
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Na-dithionite solution (5% w/v) | 1.00 | ml | ||
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Sulfur powder | 10.00 | g | ||
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NaNO3 | 1.00 | g | ||
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Distilled water | 1000.00 | ml | ||
| 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 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
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MgSO4 x 7 H2O | 3.00 | g | ||
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MnSO4 x H2O | 0.50 | g | ||
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NaCl | 1.00 | g | ||
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FeSO4 x 7 H2O | 0.10 | g | ||
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CoSO4 x 7 H2O | 0.18 | g | ||
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CaCl2 x 2 H2O | 0.10 | g | ||
|
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ZnSO4 x 7 H2O | 0.18 | g | ||
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CuSO4 x 5 H2O | 0.01 | g | ||
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AlK(SO4)2 x 12 H2O | 0.02 | g | ||
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H3BO3 | 0.01 | g | ||
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Na2MoO4 x 2 H2O | 0.01 | g | ||
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NiCl2 x 6 H2O | 0.03 | g | ||
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Na2SeO3 x 5 H2O | 0.30 | mg | ||
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Na2WO4 x 2 H2O | 0.40 | mg | ||
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Distilled water | 1000.00 | ml | ||
| 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) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
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Biotin | 20 | mg |
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Folic acid | 20 | mg |
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Pyridoxine hydrochloride | 100 | mg |
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Thiamine HCl | 50 | mg |
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Riboflavin | 50 | mg |
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Nicotinic acid | 50 | mg |
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Calcium D-(+)-pantothenate | 50 | mg |
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Vitamin B12 | 1 | mg |
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p-Aminobenzoic acid | 50 | mg |
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(DL)-alpha-Lipoic acid | 50 | mg |
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Distilled water | 1000 | ml |
Na-dithionite solution (5% w/v) #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaHCO3 | 50 | g | ||
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Na2S2O4 | 50 | g | ||
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Distilled water | 1000 | ml | ||
| 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. | |||||