|
Media
|
Component
|
Mass/Volume/Concentration
|
Comments
|
|---|---|---|---|
| Artificial seawater recipe (ASW) | Distilled water | 1 L |
|
|
|
NaCl | 27.5 g |
|
|
|
MgCl2·6H2O | 12.16 g |
|
|
|
KCl | 0.72 g |
|
|
|
NaHCO3 | 0.2 g |
|
|
|
Tris-HCl | 3.2 g |
|
|
|
CaCl2·2H2O | 1.4 g |
|
|
|
NH4Cl | 0.05 g |
|
|
|
K2HPO4·3H2O | 0.05 g |
|
|
|
Trace elements solution | 1 mL | Adjust to pH 7.3; autoclave |
|
|
Trace vitamins | 1 mL |
|
| Trace elements solution | Distilled water | 1 L |
|
|
|
Calcium pantothenate | 10 mg |
|
|
|
Niacin | 10 mg |
|
|
|
P-aminobenzoic acid | 10 mg |
|
|
|
Thiamine | 10 mg |
|
|
|
Riboflavin | 10 mg |
|
|
|
Pyridoxine | 10 mg |
|
|
|
Cobalamin | 10 mg |
|
|
|
Thioctic (a-lipoic acid) | 10 mg |
|
|
|
Folic acid | 10 mg |
|
|
|
Biotin | 10 mg | Filter sterilize; store in darkness at 4°C |
| Trace vitamins solution | Distilled water | 1 L |
|
|
|
Nitrilotriacetic acid | 1.5 g |
|
|
|
MgSO4 | 3 g |
|
|
|
MnSO4 | 0.5 g |
|
|
|
NaCl | 1 g |
|
|
|
FeSO4 | 0.1 g |
|
|
|
CaCl2 | 0.1 g |
|
|
|
CoCl2 | 0.1 g |
|
|
|
ZnSO4 | 0.1 g |
|
|
|
CuSO4 | 0.01 g |
|
|
|
AlK(SO4)2 | 0.01 g |
|
|
|
H3BO3 | 0.01 g |
|
|
|
NaMoO4 | 0.01 g |
|
| Autotrophic sulfur oxidizing bacteria media (ASOB) | ASW |
|
|
| Na2S2O3 | 10 mM |
|
|
|
|
Sodium sulfide | 100 µm | Purge with N2 mix prior to inoculating |
| Heterotrophic sulfur oxidizing bacteria media (HSOB) | ASOB |
|
|
| Dextrose | 10 mM |
|
|
| Autotrophic sulfate reducing bacteria media (ASRB) | ASW |
|
|
| Na2SO4 | 60 mM |
|
|
|
|
Sodium sulfide | 500 µm | Purge with N2 mix prior to inoculating |
| Heterotrophic sulfate reducing bacteria media (HSRB) | ASRB |
|
|
| Lactate | 10 mM |
|
|
|
|
Acetate | 10 mM |
|
| Autotrophic methanogen media (ameth) | ASW |
|
|
| Autoclave |
|
|
|
|
|
Sodium molybdate | 5 mM | (filter sterilized solution) |
|
|
Sodium sulfide | 100 µM | Purge with N2 mix prior to inoculating |
| Heterotrophic methanogen media (hmeth) | Ameth |
|
|
| Methanol | 10 mM |
|
|
| Autotrophic iron(III) reducing bacteria media (AfeRB) | ASW |
|
Autoclave |
| Poorly crystalline iron oxides |
|
Purge with N2 mix prior to inoculating | |
| Heterotrophic iron(III) reducing bacteria media (HfeRB) | AFeRB |
|
|
| Lactate | 10 mM |
|
|
|
|
Acetate | 10 mM |
|
| FeS gradient tubes for iron oxidizing bacteria |
|
Prepare FeS after Hanert, 1992 | |
| Top layer: | ASW |
|
|
|
|
Low melting agarose | 0.15% |
|
|
|
Sodium bicarbonate | 5 mM |
|
|
|
Trace elements solution | 1 mL/L | Autoclave |
|
|
Trace vitamin solution | 1 mL/L |
|
| Bottom layer: | 1:1 of FeS:ASW |
|
Autoclave |
|
|
1% high melting agarose |
|
After autoclaving, aliquot 0.75 mL of bottom layer while still hot into each gradient tube. Allow ~30 min to solidify. Aliquot 3.75 mL of the top solution and allow 3-4 hr to solidify before inoculating. |
| FeCO3 gradient tubes for iron oxidizing bacteria |
|
FeCO3 stock solution was made in advance. Add 0.1-M FeCl2·7H2O to 100 mL of sterile deionized water which had been boiled to remove O2. Subsequently, 0.1-M NaCO3 was added to the FeCl2 solution, the bottle was capped, and the headspace purged with nitrogen. | |
| Top layer: | ASW |
|
|
|
|
Low melting agarose | 0.15% |
|
|
|
Sodium bicarbonate | 5 mM | Autoclave |
|
|
Trace vitamin solution | 1 mL/L | Bubble with filter-sterilized CO2 |
| Bottom layer: | ASW |
|
|
|
|
High melting agarose | 1% | Autoclave |
|
|
Trace vitamins | 1 mL/L |
|
|
|
FeCO3 stock solution | 40 mL/L | After autoclaving, aliquot 0.75 mL of bottom layer while still hot into each gradient tube but make sure to keep it anoxic. Let solidify on ice. Aliquot 3.75 mL of top solution and bubble with filter-sterilized CO2. Let solidify while capped. |