119b .
METHANOMASSILIICOCCUS MEDIUM
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Equipment needed:
Autoclave
Centrifuge
Gassing station
Hungate tubes
Final pH:
7.6
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
KH2PO4 | 0.50 | g | ||
|
|
MgSO4 x 7 H2O | 0.40 | g | ||
|
|
NaCl | 0.40 | g | ||
|
|
NH4Cl | 0.40 | g | ||
|
|
CaCl2 x 2 H2O | 0.05 | g | ||
|
|
Trace element solution SL-10 | 1.00 | ml | ||
|
|
Selenite-tungstate solution | 1.00 | ml | ||
|
|
FeSO4 x 7 H2O solution (0.1% w/v) | 2.00 | ml | ||
|
|
Yeast extract
(OXOID) |
1.00 | g | ||
|
|
Na-acetate | 1.00 | g | ||
|
|
Na-formate | 2.00 | g | ||
|
|
Sludge fluid | 50.00 | ml | ||
|
|
Fatty acid mixture | 20.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
Methanol
(15% v/v) |
10.00 | ml | ||
|
|
NaHCO3 | 1.00 | g | ||
|
|
L-Cysteine HCl x H2O | 0.50 | g | ||
|
|
Na2S x 9 H2O | 0.50 | g | ||
|
|
Distilled water | 940.00 | ml | ||
| 1 Dissolve ingredients except methanol, bicarbonate, cysteine and sulfide. Adjust pH of medium to 7.2 and sparge with 100% N2 gas for 30 - 45 min to make it anoxic. Then dispense medium under same gas atmosphere into anoxic Hungate-type tubes to 30% v/v of their volume and autoclave. Add methanol (from 15% v/v stock solution), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use check pH of complete medium and adjust to 7.6, if necessary. | |||||
| 2 After inoculation, add sterile 80% H2 and 20% CO2 gas mixture to 0.5 bar overpressure. After growth becomes visible overpressure of 80% H2 and 20% CO2 gas mixture can be increased to 1 bar. | |||||
| 3 Note: Use 10% (v/v) as inoculum. | |||||
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
|
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. | |||||
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.5 | g |
|
|
Na2SeO3 x 5 H2O | 3.0 | mg |
|
|
Na2WO4 x 2 H2O | 4.0 | mg |
|
|
Distilled water | 1000.0 | ml |
Sludge fluid #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Yeast extract | 4 | g | ||
|
|
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 | ||
|
|
DL-2-Methylbutyric acid | 27 | ml | ||
|
|
Valeric acid | 27 | ml | ||
|
|
Isovaleric acid | 27 | ml | ||
|
|
Distilled water | 896 | ml | ||
| 1 Adjust pH to 7.5 with concentrated NaOH. | |||||
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. | |||||