119 .
METHANOBACTERIUM MEDIUM
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* Medium for Methanobrevibacter millerae DSM 16643
Supplement medium after autoclaving with 1 ml/l Wolin's vitamin solution (10x, see medium 120), 0.30 g/l DL-dithiothreitol, and 0.15 g/l coenzyme M (2-mercaptoethanesulfonic acid) from anoxic stock solutions sterilized by filtration. Omit Na2S x 9 H2O and L-Cysteine HCl x H2O.
Growth conditions: 2-3 days
anaerobic
37 °C
Equipment needed:
Autoclave
Centrifuge
Gassing station
Hungate tubes
Final pH:
6.8 - 7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.50 | g | ||
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MgSO4 x 7 H2O | 0.40 | g | ||
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NaCl | 0.40 | g | ||
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NH4Cl | 0.40 | g | ||
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CaCl2 x 2 H2O | 0.05 | g | ||
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Trace element solution SL-10 | 1.00 | ml | ||
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Yeast extract
(OXOID) |
1.00 | g | ||
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Na-acetate | 1.00 | g | ||
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Na-formate | 2.00 | g | ||
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FeSO4 x 7 H2O solution (0.1% w/v) | 2.00 | ml | ||
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Sludge fluid | 50.00 | ml | ||
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Fatty acid mixture | 20.00 | ml | ||
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Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
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NaHCO3 | 4.00 | g | ||
| L-Cysteine HCl x H2O* | 0.50 | g | |||
| Na2S x 9 H2O* | 0.50 | g | |||
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DL-Dithiothreitol | 0.30 | g | ||
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2-Mercaptoethanesulfonic acid | 0.15 | g | ||
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Wolin's vitamin solution (10x) | 1.00 | ml | ||
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Distilled water | 930.00 | ml | ||
| 1 Dissolve ingredients except bicarbonate, cysteine and sulfide. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH to 6.5 and dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary. | |||||
| 2 Note: After growth has started and the culture is becoming turbid add sterile 80% H2 and 20% CO2 gas mixture to 0.5 - 1 bar overpressure. | |||||
Sludge fluid #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Yeast extract | 4 | g | ||
|
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Sludge | 1000 | ml | ||
| 1 Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark. | |||||
Fatty acid mixture #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Isobutyric acid | 23 | ml | ||
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DL-2-Methylbutyric acid | 27 | ml | ||
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Valeric acid | 27 | ml | ||
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Isovaleric acid | 27 | ml | ||
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Distilled water | 896 | ml | ||
| 1 Adjust pH to 7.5 with concentrated NaOH. | |||||
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
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FeCl2 x 4 H2O | 1.5 | g | ||
|
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ZnCl2 | 70.0 | mg | ||
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MnCl2 x 4 H2O | 100.0 | mg | ||
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H3BO3 | 6.0 | mg | ||
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CoCl2 x 6 H2O | 190.0 | mg | ||
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CuCl2 x 2 H2O | 2.0 | mg | ||
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NiCl2 x 6 H2O | 24.0 | mg | ||
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Na2MoO4 x 2 H2O | 36.0 | mg | ||
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Distilled water | 990.0 | ml | ||
| 1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. | |||||
FeSO4 x 7 H2O solution (0.1% w/v) #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
FeSO4 x 7 H2O | 1 | g | ||
|
|
H2SO4
(0.1 N) |
1000 | ml | ||
| 1 The ferrous sulfate solution is not stable and should be freshly prepared. | |||||
Wolin's vitamin solution (10x) * #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
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 |