524 .
METHANE OXIDIZING BACTERIUM MEDIUM
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Equipment needed:
Autoclave
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Final gas composition:
2 % O2
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Final pH:
6.2
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ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
NaCl | 30.00 | g | ||
|
|
KH2PO4 | 0.14 | g | ||
|
|
CaCl2 x 2 H2O | 0.80 | g | ||
|
|
MgSO4 x 7 H2O | 3.40 | g | ||
|
|
MgCl2 x 6 H2O | 4.20 | g | ||
|
|
KCl | 0.33 | g | ||
|
|
NaNO3 | 0.25 | g | ||
|
|
NH4Cl | 0.25 | g | ||
|
|
Na2SiO3 x 9 H2O | 0.20 | g | ||
|
|
Fe2(SO4)3 x n H2O | 20.00 | mg | ||
|
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Na2SeO3 x 5 H2O | 0.50 | mg | ||
|
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NiCl2 x 6 H2O | 0.50 | mg | ||
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Trace mineral solution | 3.00 | ml | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Mix components, adjust pH to 6.0 - 6.5 and autoclave. After cooling, add the following solutions (filter-sterilized) to the medium: | |||||
|
|
Trace vitamins | 1.00 | ml | ||
|
|
NaHCO3
(5%) |
10.00 | ml | ||
|
|
CuSO4
(1 mM) |
2.00 | ml | ||
| 2 Distribute the medium into culture vessels (leave >80% volume gas phase) under a N2-CO2 (4:1) gas mixture and seal with butyl rubber stoppers. Pressurize the culture vessels to 200 kPa with methane gas. Finally add O2 gas to make up 2% O2 concentration in the gas phase. Readjust pH to 6.0 - 6.3, if necessary. | |||||
Trace mineral solution #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Nitrilotriacetic acid | 1.50 | g | ||
|
|
MnSO4 x n H2O | 0.50 | g | ||
|
|
CoSO4 x 7 H2O | 0.18 | g | ||
|
|
ZnSO4 x 7 H2O | 0.18 | g | ||
|
|
CuSO4 x 5 H2O | 0.01 | g | ||
|
|
AlK(SO4)2 | 0.02 | g | ||
|
|
H3BO3 | 0.01 | g | ||
|
|
Na2MoO4 x 2 H2O | 0.01 | g | ||
|
|
Distilled water | 1000.00 | ml | ||
| 1 Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH. Then proceed to add minerals. Adjust final pH to 7.0. | |||||
Trace vitamins #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 2.0 | mg |
|
|
Folic acid | 2.0 | mg |
|
|
Pyridoxine hydrochloride | 10.0 | mg |
|
|
Thiamine HCl | 5.0 | mg |
|
|
Riboflavin | 5.0 | mg |
|
|
Nicotinic acid | 5.0 | mg |
|
|
Calcium pantothenate | 5.0 | mg |
|
|
Vitamin B12 | 0.1 | mg |
|
|
p-Aminobenzoic acid | 5.0 | mg |
|
|
Lipoic acid | 5.0 | mg |
|
|
Distilled water | 1000.0 | ml |