| KP buffer* | 5 | ml | | MgCl2·6H2O | 0.75 | g | | CaCl2·2H2O | 0.15 | g | | NH4Cl | 0.3 | g | | NaNO3 | 0.3 | g | | NaCl | 30 | g | | Sodium acetate | 0.16 | g | | Bacto Yeast Extract (Difco) | 0.2 | g | | Trace elements solution** | 2 | ml | | Vitamin solution*** | 2 | ml | | Resazurin | 1 | mg | | Na2CO3 | 1 | g | | Cysteine-HCl | 0.35 | g | | Na2S·9H2O | 0.35 | g | | Distilled water | 1 | L | | pH unadjusted | | | | | Mix ingredients except KP buffer, vitamin solution, Na2CO3, cysteine-HCl and Na2S·9H2O. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave KP buffer, cysteine-HCl (5% solution) and Na2S·9H2O (5% solution) under a N2 atmosphere. Sterilize vitamin and 10% Na2CO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add KP buffer, vitamin, Na2CO3, cysteine-HCl and Na2S·9H2O solutions. Pressurize the inoculated vessels to 150 kPa with H2/CO2 (80/20). | | | | | | | | | | *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 |
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