| | KP buffer* | 10 | ml |
| | MgCl2·6H2O | 0.75 | g |
| | CaCl2·2H2O | 0.15 | g |
| | NH4Cl | 0.54 | g |
| | Na2S2O3 | 3.2 | g |
| | Bacto Yeast Extract (Difco) | 0.1 | g |
| | Peptone (BBL) | 1 | g |
| | Glucose | 0.2 | g |
| | Sodium succinate | 0.2 | g |
| | Trace elements solution** | 2 | ml |
| | Vitamin solution*** | 2 | ml |
| | Na2CO3 | 0.5 | g |
| | Distilled water | 1 | L |
|
| | | Mix ingredients except KP buffer and vitamin solution. Dispense the medium into suitable culture vessels under a stream of H2/CO2/O2 (75/20/5) and seal with butyl rubber stoppers. Separately autoclave KP buffer under a N2 atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically add KP buffer and vitamin solution. Pressurize the inoculated vessels to 150 kPa with H2/CO2/O2. |
| | | | | | | |
| | | *KP buffer |
| | | | KH2PO4 | 119 | g |
| | | | K2HPO4 | 21 | g |
| | | | Distilled water | 1 | L |
| | | Autoclaved under N2. |
| | | | | | | |
| | | **Trace elements solution |
| | | | Nitrilotriacetic acid (NTA) | 12.8 | g |
| | | | FeCl3·6H2O | 1.35 | g |
| | | | MnCl2·4H2O | 0.1 | g |
| | | | CoCl2·6H2O | 0.024 | g |
| | | | CaCl2·2H2O | 0.1 | g |
| | | | ZnCl2 | 0.1 | g |
| | | | CuCl2·2H2O | 0.025 | g |
| | | | H3BO3 | 0.01 | g |
| | | | Na2MoO4·2H2O | 0.024 | g |
| | | | NaCl | 1 | g |
| | | | NiCl2·6H2O | 0.12 | g |
| | | | Na2SeO4 | 0.004 | g |
| | | | Na2WO4 | 0.004 | g |
| | | | KAl(SO4)2·12H2O | 0.02 | g |
| | | | Distilled water | 1 | L |
|
| | | First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add minerals. Final pH 7.0. |
| | | | | | | |
| | | ***Vitamin solution |
| | | | Biotin | 2 | mg |
| | | | Folic acid | 2 | mg |
| | | | Pyridoxine-HCl | 10 | mg |
| | | | Thiamine-HCl | 5 | mg |
| | | | Riboflavin | 5 | mg |
| | | | Nicotinic acid | 5 | mg |
| | | | Ca-pantothenate | 5 | mg |
| | | | p-Aminobenzoic acid | 1 | mg |
| | | | Vitamin B12 | 0.01 | mg |
| | | | Distilled water | 1 | L |